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For Research Use Only. Not for use in diagnostic procedures. © Copyright 2020 by Pacific Biosciences of California, Inc. All rights reserved. PN 101-855-400 Version 03 (April 2020)
HiFi Library Preparation Using SMRTbell
Express Template Prep Kit 2.0 For De Novo
Assembly and Variant Detection ApplicationsSequel II System v8.0 / Sequel II Chemistry 2.0 / SMRT Link v8.0
HiFi SMRTbell Library Preparation Using SMRTbell
Express Template Prep Kit 2.0 For De Novo Assembly
and Variant Detection Applications
1. HiFi Sequencing Features & Applications Overview
2. HiFi Library Sample Preparation Workflow Details
3. HiFi Library Sequencing Workflow Details
4. HiFi Library Sequencing Performance Example Data
5. HiFi Sequencing Data Analysis Recommendations for Variant Detection and
de novo Assembly Applications
6. Technical Documentation & Applications Support Resources
7. Appendix
i. Optional Alternative Shearing Method (Covaris g-Tubes)
ii. Optional Alternative Size-Selection Method Using the Sage Science BluePippin System for
Variant Detection Applications
iii. Optional Alternative Size-Selection Method Using AMPure PB Size-Selection for De Novo
Assembly Applications
iv. General Recommendations for High-Molecular Weight gDNA Isolation, QC & Handling for
SMRTbell Library Construction
HiFi Sequencing Features & Applications
Overview
HiFi Sequencing Workflow Provides High Accuracy and Long Read Lengths
HIFI READS: A NEW PARADIGM IN DNA SEQUENCING
- Generate higher quality de novo genome
assemblies using a single technology
- Call all variant types – from single nucleotides
to structural variants
- Phase allele-specific haplotypes
- Identify full-length isoform transcripts – no
assembly required
- Comprehensively characterize samples with
complex variation (e.g., bacterial, viral and
cancer cell populations) to explore unique
and evolving genomes.
GENERATION OF HIFI READS BY CIRCULAR CONSENSUS
SEQUENCING (CCS)
Insert sizes up to ~25 kb
(>99% accuracy)
Longer reads allow for multiple passes of the same DNA molecule
~300-600 Gb Raw Bases / SMRT Cell 8M
~80-125 kb average RL
~1 – 4 M HiFi Reads / SMRT Cell 8M
~20 – 40 Gb HiFi Bases / SMRT Cell 8M
Average HiFi Read Accuracy: >99.9% (>Q30)
Up to ~25 kb insert size
GENERATION OF HIFI READS BY CIRCULAR CONSENSUS
SEQUENCING (CCS) (CONT.)
19,820 BP HIFI READ, PREDICTED QV: 33
>m64089_191020_002935/346/ccsGAGTCAACCGCTCTACGCACTGACTCTCGTAAGAATGAGCTTCGAGAACAAAACAAATAAAAAGAGACAGACAGCAAATTTAAAATCGGAAACCAGGAGAAAAAATGGCCAGAGAAGAGCGCAATGACAGCAACGAGGCGCTAATATAGCTATGGCGCCTCGTTACAACGTGAACTCTCCGAAATCGTCTGTTGTTCTTTTTAAAATAGTTGTTATTACATAAATAAGCGGTCGATGCCTAATACCCATTGATTTTGTAGTAAATACA
TATAATACTCGTATTATGAGTATTTACTACACAATATTTTCAACGACATGGCCGTCATTTTAATTCCGTGTCTTGGATTTATGACGTCATAAATATGATGAAAAGATATCGCTGGGTATTCCATGATTAGATTTTATGTGCGCTTTTATGATCCAAGACTCCAATAACTTCGAAGAATTCTTAGTAGAATCTAGTTATTATGAGATTAAAATAAAACTAGATAAACAACTAAGTTGTTTGAAATAATTAATTTTATTCAGCAATTTTT
GGCTTTCCAATTCAATTAGGTGCAGCCTATTAAAAACTAAGTTGCAGTACGCCACCACTTTTAGTTTTTCACAGTCTATTCCTATTATAAAGAACAAGCTGTGAATAAATTACAGTAGTTTCCCTTTCCTAAACTGAAAATACACACAATTATAATCTTATGAAAAACATAAATTCGTTTCATAAAGCTTATCGCTTTGTTTCTTTTTAGAAAATTCTCGGGCTACTTAGACCTCGGCGATGTACTCAGTACCTACCCAAATTGCTGG
ACTGGACTGCGCGATGCAATGTTCTGCGCATGTCCGTTTATATATAGGTAGCCTATTGATATTTTGTTATGACTTTCGTATGTCCTGGAGTAGCGTCCCTAGCTACACCGGCTTTTGCAGTTTTATGCACTCCTGGTAGTATATTCCAAAAAACATGTGACTGCGCCGTGCGTCAGATCGTTATGAAAGCTGATAAGCGACTGAGCGAATATGTCATTATTAAAGAAACTCCGCCATTTTGCTGCCGCCTGCTAACACTATGTGCAAT
GTGCATTCTTCTTCGATTTTCCGATTTTCATTTATGAGTGTAAAATTTTTATTTTGCGATGTAATGAAACTTCTTAATTAATTGAAATTAAACATATGTATAAAGCTGGGGAAGCTCCGTGGCGTCGTGGTTTGAACGATTTGGTTTTGATGTTAAAAAAAGCTGTTTATTTTTACATATTTTTTTATGGTATGGTGAACAATGAAGAGTTATCTATCCATCTATCTTACTTTCCCCGTGTTTAGACTTCGGAGGAACCGGTGCAACG
ATATTTACAATCATTGAAACACATTTAATACAAAAAAAAGTTCTTAAAAGAGACATTTCCTATAGGACATTTCCTGAAATATTTTGTGTAACACTAATGTTTTTATTATGCAACACAATACCTTAGCTACATATTATAAAAAAACTCACAGATATTGTATATATTTGTCTTTTACCTTATATAACGCATCTGTTTGGATTTGTACTGAAGCCATGCGTGCCAACTTTTTAGCATCAACGGTGACATGACAGCCCTTGACAGCCGTTTT
TAACGATGAAATAAAGGAAAGAAAAATTTTAATTTTATTGTGTTATGAGTTGTTATGGGCTTAAAAAACATATTATTTTTAATGGTGACGGTTAACAGCAATGAGTCATTTGGGACCCCATAAAAAAGGGTCAACTAGCCTATTAGAGTTTAATAAGATTAGTCTTGTGGTCGACAGTAATCCAAAAATACCAAAAATACAGTCCGCGTCCTAGGAATCTAGGAATTTATGTATTCAACATTAAAGGCGGATCACTACACGCCTCGAC
CCGTAAATGTGTAGCCACCGTAATGTTTGTATTTAAACACTTCGTCACATTAGAAAATAAAATAAAATAAATTGCCTATTTATTCGTATAAACAAACACATATTATTATATGTAAAATTTAAATAAAATTGCGATCATTAAAAGTTGTGTTATTGACTAGTTCCACGCAGACCAATGGTAGCGATGTTTTTCTCATATCTGCGCATGCCTGATGAGTAACAAATCAGAGGCCAATATTTACTGTTATTTATACGTTATGAATGGGAGC
GTCCGTAGCAACTGCACTTCCTTCAATACTGTGCATTCCTGGTAGTTGATGGACAAAGGCAACGTGGTTTGCACACTGGGTCACGCCCGAAGAACTATTTTATTATTTGTCCGCTGTTGAATTGGTCAGATTGTTTTATTTAGAACAAACGATCCTGTTGCCATGCACCGGGCAAAGCGTAAATTAATCAATTAGTTTTCAATTTTTTTTTCACAGGAAAAAAAATTAAAAACTATTGATTAATTACAATTTAATCTCTAACGACGTC
ATATTGATTTATTGATTCCATACTTTGTAAAACATTAAGTTAGTGTGTGTAACTTCTGCAAAGACAAAAAATAATAAATAAAAAATAAAATTCGAACTTCGAATGTAGATGGTGCACGTTTTTCTTTGGTTTCGGCCGGCCGGCGTCCGTAACAACAGAACCTACTTCTTGTACACTATGAGTCCCTGGTACCCCATCGTATAGAGAACGTGACTTAGACGGGGTTCCAAGTTTTCAACAATGTTTGCTTATTTGTTTATTTATTTAT
TTATTTGCATGTCACATAATCACTGTACAGAAATACAAAGGTCTTAAAAAATAGGATTGTATACATGTGACACCCTGCAAGGGTATAGCAATATATAATTATAATATTGGACCAGAGGGCCCTAGTTACTTAACAATTATATAAACTTATTTTTAAATTAAAATTTTATAAGCAACGTAAAATAAATTCTAATACTCGTATTACTAACAAAATTTAACAGAAACAAGAAAAACATGAGATCAAATTTTAAATTATTTACTTATATACG
TAACGTTATCTACCTTTTAATTACAAAAATTATTATATAGTGGAAGTTATTCGTTTTCGAAAAATTCCTTTAAACTGTAAAAACATTTCTCGATCAAGAACTTTTTTAAGTGCTCAGTAAAATTCGGGAGTTTCTCAAAGTTTTTGATGTCGTTTGGGATATGGTTATATATTTTTATAGACATAAAATATGGACTAGATGAGACTAATTTTAATTTTGTAAAAGGAATTTTAAGTTTATTTAAATATCTGTCATTTCTTTTGATTTT
ATTATTTGGTGAGAAAAATTCTTGATGTTTTCTGGCAAATTTACATGCTTCTAATATGTAAATGGATGTGAGTGTTAATATTCTGTGTTTGATAAAGTGAGGTCTGCATGATTCCATTTGTTCTATATTTGTCAATATTCTCAAACATCTCTTCTGCAATATAAATAATTTATCTATTTCAGAGGAGTTACCCCATAAAACGACGCCATAGGAAAGAATGGAGTATGCATAAGCATAGTATGCTGAGAGAGCTGTTTGAAAGTTTGTA
ACGCGTTTCAAGTGGTGTAGTGCATATATGAAAGATGATAATTTATGTTCTACTTTTTTAATATGCTGCTTCCAGTTTAAATGTGAATCTAGTTCAATGCCCAACAGGGTTGCTGTGTTCACTGTTTCTAATTTTATATTTTTATAACAATATTGTACCTGTAATGAACTTTTTTGATAGGGCTTAAATTGGATTAATTTGGTTTTTTTAAGGTTTAGTTGGAGATTATGTGTTTCTAGCCAGTTCGTAATGTTGTCTAAAATAAAGT
TTAATTTAGTTTTAAGTTCCGATGAGTTTGAGCAAGAGATAAGCACTGAGATGTCATCAGCAAACAATACACACAGATTATCAAAAATATCAGGTAGATTATTTATATAAATGAGGAATAAGACACATCCTAGAACGCTACCTTGAGGTATTGATCCGGTAACTTGGATGGCATTAGATCTGGTGTATGTGATCATTCCTGTCTCAAAGTCTGTATTTTGTATTTCAACGTATTTGGAATTTGGAATTATTTAGTAGTATTTGGAAGT
ATTTGGAATTGTTGAAACCAATAAATAAAACTTATAAAATCCTTATTCTACTTTCTACCATTTCGTGAAACAAACAGTAGATGGCCGATGTTTTTGTGTTCTAGGCCCACTTACAGGAAAATTAATGGAGATTACATAGTAGATATTATATCTAGACTAACCCCCTTACTAATAAAATAATAAAAATAACCGACATCCACGTGGGCGGAGCCACGGGCGACCGCAAGTTTCTTATAAATCTGTTGAAAATTTACGTCAAATGTGAAAA
TTCATGGTCTTTCTTTTATTTCTGATATTATGATGTTATTTAGTAAAGAATAATCCTCACGATGAAACCTTTCGATCGGTATGATAAAGCTACAGGTTAATTATTTTTCTCGTGTAACGGGTTCGATTCCCGGTTCAACAATGTAATTATTTAAATATCTATGAATGCAGTTTAAATAGTTTCTGAAATTGAAATAAAGTCTTCTTTCAGTAAAACGTTCTAACTGTAATATTTAAGGTACTTTCTGTGGTACTTTCCCTTCATGTGG
CATAGCCGTGGGACGGCCTGTATATTGCTGCGTAAATAAATATCTTGCTGCCCAATTAATTATTGTGCTGATCAAATAATAATGTTGCTGCCCAAATAATTATTATGCCGCCCAAATAATTATAATGCTGACAAAGTATTTGTTTGCTGCACAAATTATTTTTCGGGTTACCTTTTTGTTGCTGACCAAATAATTAAGAGACTGCCTAATTAATTACTTCCCGTTTAAAAAACCTCGGAGTAAACAGAACAAGTCCTACAAAATTGTA
TTGAACGTTCATTTTCGATTCACATTAAAGCAAAGGGGCGTTCCACTAAAAATAAAAGTGTCTACGTGATTAACTAGATGGCGCTGTCGTACACCTAAATCGTGCTATAATTTTCTCTCCTATTCTATACAATTTTCAGTTGTTGTATCATGTAAATACCCTGGTCATAAAGTATATTGAAAAAGACTATTTGTTTTATAATTTTTCACTAGATCCTCAATAAAACACCAATGGGTTGTTTAATATTAAAAATATTTACTAATAGTTG
TGAACATAAGAGCTTCCATTCCAGATTAACTACAAACATACTCTTGCAAATATGGGATCCTGGTAGCTCTCCGCAGTTTGTCAATGTGACAAAATGAGGACACTAGTTTGTAGTTTCATTGTTTCCTAGTTTATTAGGCCACGCCTAATCGTATGCCATGCCACTCCACGTACGCAGTCATTGATAACCGTCTGGTTTCGATAATGAAAACAAGGATACTATTATTTTGGATGGACCTCGGATGGATGACCAAAACTTTGTGATTTCG
ACTTCCACTGTGCTTTGGAAGCTGTTGATCTCGGTGTGTCCTGTAGGATGTCTGCTTTTTAATGTGTAATGTGAAATATAGAAACTTCGTTAGAAAACTACACAATAGAATATTGTTGACAAGAGGCTATAGAAACCTTTAAAAAGTCAAATTATTTTTAAGATAACCATAATAAGACTTTAAAAAGTTTGTACATATACGTTTCTATTCACCTGTGTCTACCCCTTCGGCGATAGAGGCGTGATTTTTATATGTTATGTTTATATTT
ATCCTTTCTATTCTACATCTCCGTATCTTCTAAAAATGAAAAGTAATTTGAATATTGAATTTTCTGATCATTGATATGAGTAAGGAGGCTTAAGGGTTTGCATAAAAAAATAATATGAATAAAGTATTATCAAAGTAAATAAACCAAGAAGTAAATCTGGCTTTTCTCTATAATTCCTTAGCTCCGTTTGATTTTCCTATTAAAATAATTAGCAGTGTAAATTGAAAGAAACTCGATAATTACATGCTACTGACAGGGTGGGAGATCG
TTAATCAAATCGCAAGAGAGGGAGCCGGAAGACGTATTTCTCTCGCTTCCACTCATCACTACCTCCATTAACTTTCTCTTTCACTCACAAAATTCGTCGCATGCAATAACAGATTCACATTTTTCACAAATCTCTAAGTTTTAGAGTTTTTTTACTTTAAATTTTCCACGTCTAGTTTTAAAGTGTGGTTTTGTATAACATGAAATAAATATGAAAGCACTAGAGATAGCCTTATGAAAAGAATATATTCGTTGCTGCAGTGAAAATT
GAACCATAACGTAATGGCTGTACATTGTATTTCGTTTGCGTGATAGGCGCAGTGTGCTTCGTAGTTAACTCGTGATGTTGCAGTGCAGTGTTCTATTCTATTGTGGTAAGGGTGCAATTTGCTTGTTCCTGCAATATGCGTGTCTTCTTGTGGGTTATAGGAGCCTGGTAGGTGATGCCCTTTTACACGTGACGTCACCCCGAAATCCCCATCCGCTGTCCCGTTTTCTCTTAATGGAGGGTGCTTTTTCTTTCAAACAATGCAATGT
GAGGAAAGCAGTTTTATGCCGCTTTTGGATTACGGATTTCTAACTACTTACTAACATATTGGGTCCTTACCTATGAAATTGGTGTTTTTTTTCTCTCTTAGTATTTTGTCTGTAACTATTAAGATTCAGAGTATTCCTAAAAAATAATTACCTCGTCTAAAAATAGACTATATTTTTGAGCTCTTTTGATTTGTTTTGATAATTTGTTGTAAGTTAATTGTGTTAAAATTCGACACGGAGACAACTCTTTTTAAGTGCCTCCCGAATA
CATCATTTTATTTGAATTCACTTAAAAACGATAAATAATTTTTATGAAGTGTGATATTACACATATTACTAGAGCTTGTCTAATGATACATGCCAAATATTATTATCTTTGGATTTGTCGTTGTGAAGAAAACTGAAAAAACTTTAGCATGGAAAATGCAGAATTCCCCAAAATCTCCATACAAAACACCAATTTCATAGGTAAGGACCTAATATTAATACCGCATATTAATTACTAACACCCTTACTAATAAACAAGGTATAAGGTT
GAAATAGTTATTCAGTGTTTTGTCTTCGTCAGACATTCAGAATACAGTGGTTGTCAGAAAGTGACAAAACACTGAATAACTATTCCTTCCTTATAACTCGTTTATTAGTAACGGGGTTAGAATATTTAGAATGTGTCCCAATACCTAACTTTTCATCACACAAACATTTGCCTCGACTTGGCTTTGAACCCAGAATCGCTAGAATCGCTTCCATTCTTTCACGGAGGTCGATTCCCAATAAACTCAAAACCGAACGTTGAGATTACCA
TTTTCAAAGAATGGAAGGGTTTTTGTTGATAAAGTTGAAGGTTTTCATTCATTTTTTAAATAAACGTTTATTTTCAATAAAACTAACGCATAAATAAGTGGTCATGAAGTAGAACATTTTGCGGCACAGCTTCAGTACTACGAATTATGCATGTTCTGAAAACATCTTATACAATAGGTTTTTTTTGTAATAGAAGAATGTCTATACTCACTGCCAAAAAAACAACACATGATAAGTATTACTAGTACTAGACCAATGAAAATAAATT
CCCCAATATGGAAATATGTTTCCATATTTTGAAGTGGCTCTCCGAGATTCCTTTGTGTCGTCAATAGAGTGCTCATGTTAGATTACTGAATGGTCTGTAATAACGCGTATAGAAAAGTTTTGTTTGTTGTTCCAGGATGGGCTAGCGCCCCACTCTCACTATATGGTTATGATAAGCTATATGTATGTTTACGATATGTAGTTATTTATACGTCGGCCGTAACGTTACTAGCCAGATCTTTGTGCATGTTATGTTGTTGATTGTGACT
GAGTAGGTTAAAAAAATTTTTGTTATGGTTTTTCAAAATTTCTTATAGCAGAATGTCAAATACACTATTGGATGCTACTATGGCTCTACCTTTAACTATTTTGTATGTATACATATGTATTCATATTTCCTATTATAATTATATTTCTTTTCTTGTTTTTTTACATAATTTTTACGTTGTCAATTATAGCTGTTAGGTATCGGAGATAAATAAGAAGCCGAGCTTCCGTCTAACAGTTTTTAATAATTAGTAATAAAAAATCAATGTC
AAAAATGGCAAGAAAAGAATATAAGTCAAAAATTAAAGAAAACATAAATGTATCAAAAAGTATGCTACCTACAATGTGTCTATGGCCAGATCTGTAGCCAACAGAACTACCCCCCAGTGAACGCACGAATTCAGGGTGGCGTACAGGTACTGGCCAAGGACACCAGAAGCCGGGTCGCCCGCTTAAACGACCCCGCTCCAGTGGCAACATCGACGACTCTAACTATGCCATCTCGCCCGGGGTACAGTTTTGTGATCCGGCCGCGAGG
CCACGAACCACGGGGGAGGGTCCCATCAACGATTAGAACCAGATCTCCAACTTTAAAGTGCCGGCTTTGCCCAGGCTTCCGCGGGTAGAGTGTAGGCAAGAACTCCTTAGTCCACCTCTGCCAAAAGTGGTCGGCGAGACGGAGGCCCTTTTTGAAGGATAGCCGACCAAATAAATCAGAGTCAGAGAAAGTTGAGAGAGGTAAATCATTGACGGGACCCAAGAGAATGAAATGAAAAGGCGTGAGCGCCTCCGGCTCTCCTGGCTCC
ACGGAAACATGAGTTAGTGGCCGGCTGTTCACAATGTTTTCCACCTCCAGAAGCAGCGTGTGCAGGACCTCTTCTCTGGGAGAGCGCTCCTTCAGGGTCACAGCCAACGAGGTCTTGACGGTTCTCACAAGCCGCTCCCAGGATCCTCCCATAAATGGGGCTGCAGGAGGGATGAAGACCCACCTGATTTTCCTGTCAGCGCTGAACTCGTAAATAGCAGGTAACAAGCGCGCAGCGCCGACGAAAGCTGTTCCATTGTCTGAGTAGA
TGGACTCCGGGCACCCTCGACGAGCGATAAACCTGCGAATCGCCATAATCGCAGAGTCTGAGGAAAGCGACTCTACCACTTCGAGATGTACTGCTCGGACTGTCAGGCAGGTAAACAACGCTACATAGCGTTTCAAATGGCGCCTTCCTTGCGCAATCGTGACTGGCCCGAAGTAGTCCACTCCAACATGAGAAAACGGCCTCGATTTATAAGCTAGGCGTGCCGGTGGTAGGTTTCCGAAGGGTGGTGTCACTGCTCGGGCTTTCCG
AATACGGCAAAGATTGCATCTGGATAAAACAAGCTTCACCGATGGTCGCAGCCGAAGAATGAAAAACCTTACTCGTAGCGCGTTGACCACAGTCTCAACTCCGCTATGCGCCATCTTTCGGTGGTAGTCTCCGATCAGCAATTGCACTATTGAATGTCGACCGTCCAAGACAATTGGCTCCTGCTCATGGCCTGGTGGCAAAACTTCTGTTAGGGAGACCCTGATAGAGATCCGGAGGAGATGATCAGACCTAACAATGAGTGATACA
TTTTTAAGCCTACTGTACCTGGGGAGTGGCATAGAATTTCTAACGCATGTCATTTCCTCTGCCAAACTATCAACTTGAGACTTAAGCAACAATTGGCTTTCGGCTCTAATTACATTATCTGCCGAAAGTAAACCCGGCATTTGGGATGGACGCTTTAAGAACACGTTCGCGCAAAAATAAATACGGGCAGTTGCACGTAAAAGGCGTGTCCACGAGCTGAATTTCGTAAAATCTGCAACTACGGGTACAATAGAAATTAGTACTTGAC
TTTCAGACGAACAATGAACCATTTCAGGCAAAGGAAATGACACTTGGGGTTCCTTGGGCCAATCAGAATTAGAAGCCAGGAATGATGGCCCTAAGAACCAGCGGGAAATTTTGAATTTGTTGTCTGGTCTCGTAGCGTCGTCCGCCACATTTTGTGCTGAAGTCACATAATGCCAATCCGAGACATTAGTTATTTCAGTAATTTCCCCAACTCTAAGAGCCACAAAGGCTTTCAGTGAGCGGGCGTCGCTACGTATCCATCTTAAAAC
GGTCAAACTATCAGTCCAAAAGTAAATTTTGAAGGTTTTCGTCGGTGACAGTTTAGAATGTAGGCACCGAGACGAGCAGCGATTAACGCAGCTTGCAATTCTAACCGCGGAATTGTCGTTAGTTTGAGCGGTGCTGAGCGAGTCTTTCCGGCGATCAACGATATCTTAAAAGTCTGATCTGAATACACAAAACGCCAGTATGCCACACACGAATAGGCAAGCTCGCTTGCATCCGCAAAAACGTGGAGCTCAACCTCTGAAAAGGCAG
AGTCGCTGAAATAGCAGCGAGGTATATTGACATGTGCAACATCTTCAAGGTCTTTAAACCAAACGTACCACAGGGCTGATAGACTTGAAGGCAGCTCCGCGTCCCAATCAGAGACGGCCTTCCAAGTCTTTTGAAAAAGTATTTTACCCTTGACTGTAATAGGACTTAGGAGACCTAGAGGATCATAGACGCTCATCAACCTTGACAATACATATCTCTTTGTTAGGATGCGAGGGACAGTTACAGTTAAATTTCGCGTAGGGTGAAT
AGTATCAAGTGTTGTATTCCAAGAGACTCCTAAAACCTTAACGCTGTCATCTGAACGCGTGGCACGCAACTCTCCCGGAACCAATGACAAAGCGCCCTCTACGTTAGAAACCCAGGACCTCATCTCAAAACCCGCACGAGAATGAACGTCAACGACATCCCTAGCCAGATGAGCCGCCTCAGAAACATCATTAACCGACGTTACGAGATCATCCATATAATGGTCCTGAATGATTACATTAGCGGCTTTCGGAAACCTGTCCTTATGC
TGCTCAGCATTCAGGTTCATTAGATAGAGAGCCGTGAAAGGACTCGAGGTAGCGCCAAAAATCATGGACGACATTCGGTAATGACATAAGGGCTGAGACGGATCAGCGCGCCACAGAAACCGCTGAGCGTCACGATCTAACATGCGGATTTTTATCTGAGGGTACATCTCCCTAATATCGGCGTTTAGGGCTATCAGTCCTTCTCGAAAACGCATAAGGACCTCAAGAAGGGGTTGAAGTAGGTCGGGCCCTGACAACAAAAGGGAGT
TTAAGCTTACTCCTTGAGCCTTTGCTGCGCAATCGTGAACAACCCTAAGCTTTTTCTTCTGTCTATGGACAACACCAAAATGTGGCAAATACCATTTTACCGGAGTATCAGCGTGTGGCATCTCGCACTTCTCAGCATAACCCTTGTCTATCATGGCTTGAATATTACTACGATACTGAGTTGCATACTCTGGGTCCTTAGCCATCTTTTGTTCTAGAGAAGTAAATCTGCGACGTGCTAGAGCGTAACTATTAGGAAGCTGTCCCAA
ATTAACATCTGACCGCCATAGTAGCCCTGTCTCAAATCGACCAGAAGGGAGTCTGCGTGTGTTGACACGAAGGGTTTCTATGGCGAAAATATCATCCTTGTCAAGTCGCTCTCTCCTATCAATACCAATTGACTCTAGTTTAAAATTTTCTCGAATAAGATTCTCTAGAGAATTATCTCGTACGTGATTGACAGTAGAGTCTGACCGGAAAACTCTACTACAGACGTTCTACTTGTCGGGAAACCGTAAACTACCCATCCTAAAGGAG
TACGACATGCCACAGGCTGAGCGCGTGAGATGCGCCTAACTTCCGACGCTACCAGCAGGTGCCAATTGTCCATGCCGACTAGAATTGATGGTTCCACGTCCGAGTAACTGCACAAATTAAGACCGTTAAAATGCTCCAAATTAGTATCGCATCTCGATAAAGACTGACGAGAGAGCCCAAGATCGCGAACGGAACGCACATGTTTCAAAACATGTGGTTGACAAGTCTTATCATGACCTTTAATTGTAAAGCTAACATACTCCACGTC
TACTCGCTTTTCTAGATTACGCACACATGAGATGATAACACTCTTTTTTAGCCCCGTAACACCGATAGCAGACGAAACCTTAGCGTCTATGAACGTAGCAGTTGAACCCTCATCCAGAAGGGCAAAGGAATCTATGCTGCCACTCGGACCACTGACGGTTACCGGCAAAATCTTAAGAAGTGGTCTGGAATCTTGCTGCTTACATTGATCGCCATCTGTTTCCAAAACTGTCTGCCCCAACGAGTAAACCTGGGTTGAGGAGTCGACT
TTAGACCTATCGTGCAACGCATCGAGTTCGGTAGTCGGTTCTGCAGAAAATTGAGAACCTACAACAGGATCGACATTGGTAGCTGCTTCATGCAAGAGAGAATGGTGGTGACGTTTACGTCGGCAACCGACGACCTTACAACATTTCTTGCAGTTTCTATAGCTGTGACCACCTTTCCTCAAGCATTTGAAGCAAATAAAATTGTCTCTTACCCAACTCAATCGTTTTTCTGCAACCAAATCACGAAACATCTGACACTTCTGGATTT
CATGCGTAGCCTTACAATAAGCACAACCAGGTTCGCTAGAGGTGTTATCTGTGTGACTTGGAAACGATGACTTAGGAACTTTACCTGGTTGTTCGAAGCCTTCTTTGTGTCTCTTGACAGAGCGAAGATGTCCATAATTAGAGACAGGCCTTCCAGTCAAAGTCGTTTGACAGACTGTAGAGGGGTGCTGGTCAAGAGTTGCTGAAGTGAGGGAGGAGTCGACGAAGTCAGCCTGGACAAAATGAGCATGAGACCTATCTGACTTAGG
TTTAAAATCTGTCAGGATACCATATCTTAAGTGCAATAAAGCCTCATGCGATAAGAAATCAGAAAGCTTTTCCAAGCGAAACTTATTTTCATGCTCTTTCTGAAAGGCAAACGCCAACCACTTCTCCCTCAGGAGAGGAGTAAGCTTTCCAAGATAACGTTGCACAGTTCAGGGTTAGCTAAATGGACTTGCTGCTGCAGAAGCTGAACGGTACTTATGCAGTTGCGTACCTTGCATGCAAAAACATTTAGCTCCTGTGCATCGTACC
CGCTTAACAGCGTAGCCGCTTCTGCGCAAGAACGGAAGAACATTTGGTGGCGACTGAATTACCGCTTGAGGAACTCACCTGCGCCCTTTGCTTCGAACGTGACGTGGATGGGGACGGCGGCGGGCCCAACTCTGCGACCGCCGCGGCGGCATTATCCGTAGCGGAGTTAGCCTGCTGCTTTGCGATAGCACGGGTGATCATGCCGCGAACTTTTAAATAACTTCTGACACCAGAAACTGTTAGGTATCGGAGATAAATAAGAAGCCGA
GCTTCCGTCTAACAGTTTTTAATAATTAGTAATAAAAAATCAATGTCAAAAATGGCAAGAAAAGAATATAAGTCAAAAATTAAAGAAAACATAAATGTATCAAAAAGTATGCTACCTACAATGTGTCTATGGCCAGATCTGTAGCCAACAATAGCTATTAATTTTTTTGTTGAATAAAATACTACACATCTACTCTTACCGCGAATCGGACCCTCAGATATGGTCACGAAGGTCGTAAATATTTTTAAGACGTTGTTTCAAACCATCG
CGTTTGAATATAACGTATCATCGTTTGACACCCTTCGGAGAAAAATATTTGCCCAGCAGTGGGAGTCTTTCGACTGAGATGTATACCATGGCTGAAAAAAAGATACTGAATAAGAACACTCGAAAAAAAAAATCGGCATTCGGCGATCTTAACGCTATGCAGCAGTCTCTTTCAGAAAAACTGCATCGTAATAATACTCGTATAATAGAAAAAATCAATCGTTACACAGTTATTTTCAAGAACATTTGTATCATATATTTGTAAATAT
TCTTTTTTATATTGCAAGATCATTTCTTAGTTATTGTTAAATGGTTGTTCCTTTGCGACCCATTCAATGTCTGCGGTCACCAGTCGACCGTTGTTGACAGCGTGTCAGACTGAGAAGGGTCTATCGTTGGAATAACTGACATATTAACACAGTACTTGTACACATGGAAACGATTTTATATAATACGACTAGAATTCTATTGTGATAGTTTTTTTGTTGATGCTAATGAATACCTGGCAGTATCTATAGACGTAGTTACAATTAACCG
TATTTTAACAGAATTAAATTCAAATAAATAAGTGTGCCTATTAGTATTTTTATTAGTTACTAGTGGCTTACCGCGGTTTCACCCGCGCGGTACCCGTTCCAATGGGAATATCGGGATAAAAAGTAGCCTGTATTCTGTATACCAAATTTCAATTAAATTGGTTCATAGTTTTGACGTGAAAGCTAGACTGACAGATAGAGAAATATACAGACAGAGTTACTTTCGCATTTATTATTAAGTATGGATTGTTATTATTTTATTTTAATTT
ATTTACTTGGAAGAATCGTTCACTCAGGTTGAACGTAGAACCCTAATGGACCATCGATTTGTAAAATAATAATAATAATAATATCCTCAGACATTCACACCGATCAACTAGCCCCATTTTAAGCAACTAATGCTGCTTGTGTTACGGACACTAGATGACGGATAAATATATTTAATAGATAATAATAATAATAATAATAATAATAATTTATTTCGATAACTTAGTCTACAATTACAAATTAAATGTGGGATCTCCCTTTTAGGCAAGA
CATCGCCTATAGATAAATACATATAAATAACCAGAATATATAGAAATATATATAAACATCCAAGACTGGAGAACAAATGCCTGAAAATTCATCACCCAAACATTTGCCCCCACTGGGGATCGAACCCAGAACCTCTCGAGATGGCGACGCCTTGAGACCGGCATACAGACCACTCGACCACGGAGGTCGTCAAAAGAACGAGGAAATGCGTTGTATGCGGGCTGCACAGGACCCACCTGCTTAGAGAATTGGAGATCCTTATGGGAGG
CTTGTATTGTATCCAACAGTAGGTGTCTGTCACCTGATATGACATATTGGCGAGTTTAGGTCATTACCTTGGTTTTACATACAAATAACGTATACTCTAAGGTTCCTGAATCTATATAATTTAATTATGAAAATTATCGCATTCGTCTCTGGAATAACCTTCCATTACCAATCAAACAATTCCAATCCTTAGCATCCTTTAATGATCTGGTCCATACCCATTTCCTTCATCTTTGACTCCTTCCTCCAACACACATTGCAGTTTTCGC
CTTTTCTTTGTTGCTTTTTGTGTAAGAATATAGATAATAAGAATATATTGCTAAATAAAATATTTATACAGGATATTGTAGCATTTATAATATAGTATGTATTTTATGTATGTATATAATATATTATGTAATTTATATGTAGTAGTTTATATTTGCACCTTATATATGTTACCTACCTTTTCTTTCATTCTATCTCTGTCCAAAGGTTGTCTGGAAGATATTGCTATTAGCTATAAGGCCGCCTGTGTTGTATTGTATTTTTTTACTA
TATTTCTGTTTTAATTAGTTGCAATAAAGAATTTTATCTATCTATCTATTGCCGATAACTTTCATGAAGAATTTTCCTATTCATGAATACCATGAACAACAAACCTAAACTCACCCTTCTCACTCAAGTTAACTTATGCCAACTATAAATAATAAAAATAAACGAAACCCACATTATGATTGTGTAGTAAAATCTGACAGTAAATTAATATTTCTATTTGGTAAGGCCGGTACATTTAAAGCGTTTTGTTTTAAAAATACAGCTGGCT
GTGTGCCAAGCAAGCGAATACTATATTGGGCCAGCCTGGCTAATTGTTACTTCTGAATGCAACGAGGGAAATTTCTACAGCCTCCGACAACATTGTGGATAATTTTTCGACTGTATCCAACGTTGTAGTTCCTTTACAAGAATACAAAAAGCTCTTTAAACCTATTATGAGTCCTGTGCGCATTTCGGAATTTCGAGTTGCGTTCGAGTTTTTCACTTGTTTTACCAGGAAGTTATTGTAGGGATGACTGTAATTATGATACAAACAT
TTTTGATGATGTTGTAAAAAAAAAGCTATTGCATAGCATTTTGTATCAACTTATGCAATTATAATTTTATTTATTTATTTTATTCATGCAGTATTTCTTTTTCTTTTTTATTATTTTACAATACATGTGGGTTTTATTATCTTAACGCCGTGTCGAAGACTGCCGAACTGAAACGACGTTAGACGATGCACGGCCTTCAAGCCACACCTCCTTGCGCATCGGAGTGGGGAGCGTGAGGTTTTTTCGTTACGGAATTTCTGGATTCGGT
CCCCGCGCTCAAGGCCCGAGATAGAAGCTATGCAATAGCTTAAAAGCTTGTAGAGAAAGAGATAAAACGCCTAAAAATGGAGACCTAACATATCAGCAAAATATGGGTGATAACACCTCCCTACCCCTTTGCAGAAAGAGGCGCGATGATATGTTATTCCATATTTTTTTTACATATTTACATGCAGTGTTATTGTAAATGGACAACCTCCGTAGCCGAAATTTGTACGCTGGTTCGAAGGAATCTCGTCATTTTTAGAGGTAGGCTC
ATTTGATCTTCGGTGATTTATTCTCTAAAACTTATATGTATTGAATATTATGGGCCCTGGCAGGGAAAGTACCTTACATATTTAAAATAGATAAATTTACATAAAGGACTAACTTTTGTTTTTAATAATTAACATAACTGATCTCTCAGTTTTTTTTTAAAAAAGCTTACCCAAACCTTCCCTGGAATATAAAATAATTGTTAGACATGTTTAAAACGTAAAGCTTAATAAAGAAATTAAATTTTCAACAATGACTTAATCGGTTAGT
GGAATTAAATCGCGAACTTTTTTTATTCATTTTTTCCTTATTCGATTCTCGGTTTGAGTAAGCTAGCTAGCTATTTTGAGAATATTTAGAACTTAGTTATGTTTCTTATTCAAAGTAGAAGTATGCTCTTGTGTAAATTATATTTTTTTTTAATATTAGCTAATAGCTCCAGGGCCCACAAAATTTTCCACTCTGTATGTAGGTATATACTTATCTATTACGCTCATTAAAACTTATCCGTCATTTAGTATCCATAACACAAGCAGTA
TAAGCTGCTTATTTTAGGCTACTTGATAGGTGAAAAAAATATGTATTACATTCATACATACATTCACGCCTCTTTCCCAGGGGGGTTGGCAGAGATTACAGATCTGCACTTGCTACGATCCTGCATCCCGCACATGACACACACTGTCTCTACCCTCATTACTAACTTTATACATGCCCTTCGGTTAATTTTAATAATTATTTATTAGAATATTAAGCTTTATTATGATTAATAGCGATGGCAATTCGTTAGCTTATAGGCGCCATTT
TCTTTATCCTCTACTTAAGTAGTAGCCAGCAATTTTAGGGCAGTGACCTAAGGTCAACGACTTGTAAAGGATAGTTCTTATGTTTTAAGAATACCGCATAGTAGATACGAATAACGTTGCAGTCTAACCGTGCAGAAGATTTTTGTCTTTCCGGATGATTAGATGTTATGAATATGAGGGAAAATATGCTTTAAGTATGAAGACTATAAGGTCTAGTATTTAATTTGATGTTATAGTTATCTTCGCACTGCTCTATAGATTCTTTTAT
ATAAAAATGACTTTTATTTTTTTATTAACAGCAACATTCTAATCTAAAATAATATAGACCAATTTTATATATTTACTTTTATATTTACCTGTTAGATTGAGACTGAGAAATATCAGTCGAATGGGAGTGTTTAAAACGCCTGTCTACCCCTTTGGGATAGAGGCGTACGTTTATGGTTTATTTTGATGTTTGTTGTATGACATATTACACTAAATCTCTACAAAGAAGAACAAGACGAGCCGAGTTGCCATGCATCGGGCACAAAGTG
AAGCAAGCAATATTAACAATGAACTGTACATAACTTTTAATTCAAACTCGGTAGAACTCGCGAATCCCGCGGCCTCTCCAAGGAGTAGTGCCCTCTGTCCCAATTCAGCTAAGGAATAGTCAACGTAATTACTAAAGCAGACTTATTGTCTCCACCACATGTAAACCGTTTGAATCATGCATTAATAAAACTTCATGTCCCCAACAGTGGGCACTGGCTTTTACCTGACTTCATATCTTACACATTTATATCCTTAGAGGAGTATACC
GAGGGGTCATGATTTTTGTAATCTAACAATGTATGTTTGTATTTTAAATTGATAATCCAGACTTATTATAAATGCGAAAGTAACTCTGTCTGTCTGTCTCGCTTTCACGTCAAAACTACTGAACCAATTTAACCGAAATTTAGTACACAGATAGTCTAGAACCTGAGAAAGGACATAGGCTACCATTTATCCCGATATTACCATGGGAACGGGTGCCATAGGGGTAAATGTAAAAACATTTAAATAACGCCTCCCAGAAAAATGAAGG
ACAAATTTACGCCACTATGAACTTTGCTTCTAATAATAGAGTAGGAGTAAATTTTCAATTCCACCAATTGAGGCTACGGTTCTTATTTTAAAAAATCTTTCAGTGTTCAATAGCCTATTTTTTGAGGAAGGCTATAGGCTACATACCATTAATCACACTATCTCAAAGTCACTATTCTACGCGGATGAAGTTGCGGGCAAAAGCTAGTTGATAAATTCATTGATGTATTGATAATTCATAGACCATCAAGTCATTTAAAAACGTAATA
AAAAAAAATGTGTTAAAAGCCTCAAAAAAAATTATATAATTTTTTATGCTGGTGGCTAACAGAAATGAGTAATTTGGGGCCCAATAAAAAAAGGGTCAACTAGCCTAATATTTTAATTTCTTTTTTGTACTCATTATATTAGGTCCTTTCCTATGAAATTGGTGTTTTGTATGGAGATTTTGGGGATGTCTGCATTTTAAATACTAACATTTTATCAGTTTTCTTCACAACGACTAATCCAGAGATAATAATATTTGGCATGTATTAT
AAGACAAGCTTTAGCAATATGTGTGTGAACACGCTTGATAAAAATTATTCATCGTTTTTATGTTAATGCAAATCAAATGATGTATTCGGGAGGCAGTTAACGTTTAAACATCAGCCTATGTTTGTCCACTGCTGGACATAGGCTTACCCTAATGCATCGAGAGGTTATTCGAAAGATTAATTGCTGTTTAGCTGAAAGGTAAATTGTGAAATCAATCTAGTTTCAAGAAATTAACTGTTTATCCTATGTGGAGTTATCAGTAAAATGT
TTTTCCACTAAATTGTTAGTTAAAATTTTACACAGCAATCCAACATAACTCTTCTAGTCATAGCAGCAGAGTGGTTTTTTAAAAACCGACCGTTTGTTGCCGACACCACACCTGTTTCGCAATTTTTACAACATTCAAAACACCACAAACTCATTTAAACTACACTACATCAAATTGTCACGTTAAATTTATTACAATAGAGTTGCTTTTTGTTACAAAAACCTTTAAAAATGTATAGTTTTACGATATTGAACTTTAAAGCGGTAAT
AATAAAGCCTTAATATTGTGTTTTAAGACTATTATCTAACGACGCCGTTCGCGTAATTTTAGTTACATTGATCCAATTAATATGAAAACTTATAGCTGCTATCGTGTTTCGACACTAGATTTTCTTTGACACCAAAAAAGTACTAAGCTTATTACATAAAAGGCCAAATAGTGTTATAATTGTTTGTTTTGACAATCACTGATGAAACTTTGATATATTTTTCCCGATGTAGGTATTCGTCTTAGTACACTACTTAAATAAATAGAAC
ATTTTACTAAAATGTTCTTCATTGTAGTAATTATCGCTATTTCTTTTGAATCAAAAATTTAATAATATTAACGCGACGTTTCCCGATCACGCTATATTCCCGAATGGGTAGTTTTAATCTGAGCAACTTTACAATAATAACTTTTTAAATCATAGTTGATTAATAAATAAATTATTCAAAAAAAAATACAAATTTAAGACTAAAAATACTTAAGACAAAATTATACTTACAGACTTTTTATTATCATTT
19,820 BP HIFI READ, PREDICTED QV: 3319,812 bp correct, 8 errors
99.96% accurate (QV34)
>m64089_191020_002935/346/ccsGAGTCAACCGCTCTACGCACTGACTCTCGTAAGAATGAGCTTCGAGAACAAAACAAATAAAAAGAGACAGACAGCAAATTTAAAATCGGAAACCAGGAGAAAAAATGGCCAGAGAAGAGCGCAATGACAGCAACGAGGCGCTAATATAGCTATGGCGCCTCGTTACAACGTGAACTCTCCGAAATCGTCTGTTGTTCTTTTTAAAATAGTTGTTATTACATAAATAAGCGGTCGATGCCTAATACCCATTGATTTTGTAGTAAATACA
TATAATACTCGTATTATGAGTATTTACTACACAATATTTTCAACGACATGGCCGTCATTTTAATTCCGTGTCTTGGATTTATGACGTCATAAATATGATGAAAAGATATCGCTGGGTATTCCATGATTAGATTTTATGTGCGCTTTTATGATCCAAGACTCCAATAACTTCGAAGAATTCTTAGTAGAATCTAGTTATTATGAGATTAAAATAAAACTAGATAAACAACTAAGTTGTTTGAAATAATTAATTTTATTCAGCAATTTTT
GGCTTTCCAATTCAATTAGGTGCAGCCTATTAAAAACTAAGTTGCAGTACGCCACCACTTTTAGTTTTTCACAGTCTATTCCTATTATAAAGAACAAGCTGTGAATAAATTACAGTAGTTTCCCTTTCCTAAACTGAAAATACACACAATTATAATCTTATGAAAAACATAAATTCGTTTCATAAAGCTTATCGCTTTGTTTCTTTTTAGAAAATTCTCGGGCTACTTAGACCTCGGCGATGTACTCAGTACCTACCCAAATTGCTGG
ACTGGACTGCGCGATGCAATGTTCTGCGCATGTCCGTTTATATATAGGTAGCCTATTGATATTTTGTTATGACTTTCGTATGTCCTGGAGTAGCGTCCCTAGCTACACCGGCTTTTGCAGTTTTATGCACTCCTGGTAGTATATTCCAAAAAACATGTGACTGCGCCGTGCGTCAGATCGTTATGAAAGCTGATAAGCGACTGAGCGAATATGTCATTATTAAAGAAACTCCGCCATTTTGCTGCCGCCTGCTAACACTATGTGCAAT
GTGCATTCTTCTTCGATTTTCCGATTTTCATTTATGAGTGTAAAATTTTTATTTTGCGATGTAATGAAACTTCTTAATTAATTGAAATTAAACATATGTATAAAGCTGGGGAAGCTCCGTGGCGTCGTGGTTTGAACGATTTGGTTTTGATGTTAAAAAAAGCTGTTTATTTTTACATATTTTTTTATGGTATGGTGAACAATGAAGAGTTATCTATCCATCTATCTTACTTTCCCCGTGTTTAGACTTCGGAGGAACCGGTGCAACG
ATATTTACAATCATTGAAACACATTTAATACAAAAAAAAGTTCTTAAAAGAGACATTTCCTATAGGACATTTCCTGAAATATTTTGTGTAACACTAATGTTTTTATTATGCAACACAATACCTTAGCTACATATTATAAAAAAACTCACAGATATTGTATATATTTGTCTTTTACCTTATATAACGCATCTGTTTGGATTTGTACTGAAGCCATGCGTGCCAACTTTTTAGCATCAACGGTGACATGACAGCCCTTGACAGCCGTTTT
TAACGATGAAATAAAGGAAAGAAAAATTTTAATTTTATTGTGTTATGAGTTGTTATGGGCTTAAAAAACATATTATTTTTAATGGTGACGGTTAACAGCAATGAGTCATTTGGGACCCCATAAAAAAGGGTCAACTAGCCTATTAGAGTTTAATAAGATTAGTCTTGTGGTCGACAGTAATCCAAAAATACCAAAAATACAGTCCGCGTCCTAGGAATCTAGGAATTTATGTATTCAACATTAAAGGCGGATCACTACACGCCTCGAC
CCGTAAATGTGTAGCCACCGTAATGTTTGTATTTAAACACTTCGTCACATTAGAAAATAAAATAAAATAAATTGCCTATTTATTCGTATAAACAAACACATATTATTATATGTAAAATTTAAATAAAATTGCGATCATTAAAAGTTGTGTTATTGACTAGTTCCACGCAGACCAATGGTAGCGATGTTTTTCTCATATCTGCGCATGCCTGATGAGTAACAAATCAGAGGCCAATATTTACTGTTATTTATACGTTATGAATGGGAGC
GTCCGTAGCAACTGCACTTCCTTCAATACTGTGCATTCCTGGTAGTTGATGGACAAAGGCAACGTGGTTTGCACACTGGGTCACGCCCGAAGAACTATTTTATTATTTGTCCGCTGTTGAATTGGTCAGATTGTTTTATTTAGAACAAACGATCCTGTTGCCATGCACCGGGCAAAGCGTAAATTAATCAATTAGTTTTCAATTTTTTTTTCACAGGAAAAAAAATTAAAAACTATTGATTAATTACAATTTAATCTCTAACGACGTC
ATATTGATTTATTGATTCCATACTTTGTAAAACATTAAGTTAGTGTGTGTAACTTCTGCAAAGACAAAAAATAATAAATAAAAAATAAAATTCGAACTTCGAATGTAGATGGTGCACGTTTTTCTTTGGTTTCGGCCGGCCGGCGTCCGTAACAACAGAACCTACTTCTTGTACACTATGAGTCCCTGGTACCCCATCGTATAGAGAACGTGACTTAGACGGGGTTCCAAGTTTTCAACAATGTTTGCTTATTTGTTTATTTATTTAT
TTATTTGCATGTCACATAATCACTGTACAGAAATACAAAGGTCTTAAAAAATAGGATTGTATACATGTGACACCCTGCAAGGGTATAGCAATATATAATTATAATATTGGACCAGAGGGCCCTAGTTACTTAACAATTATATAAACTTATTTTTAAATTAAAATTTTATAAGCAACGTAAAATAAATTCTAATACTCGTATTACTAACAAAATTTAACAGAAACAAGAAAAACATGAGATCAAATTTTAAATTATTTACTTATATACG
TAACGTTATCTACCTTTTAATTACAAAAATTATTATATAGTGGAAGTTATTCGTTTTCGAAAAATTCCTTTAAACTGTAAAAACATTTCTCGATCAAGAACTTTTTTAAGTGCTCAGTAAAATTCGGGAGTTTCTCAAAGTTTTTGATGTCGTTTGGGATATGGTTATATATTTTTATAGACATAAAATATGGACTAGATGAGACTAATTTTAATTTTGTAAAAGGAATTTTAAGTTTATTTAAATATCTGTCATTTCTTTTGATTTT
ATTATTTGGTGAGAAAAATTCTTGATGTTTTCTGGCAAATTTACATGCTTCTAATATGTAAATGGATGTGAGTGTTAATATTCTGTGTTTGATAAAGTGAGGTCTGCATGATTCCATTTGTTCTATATTTGTCAATATTCTCAAACATCTCTTCTGCAATATAAATAATTTATCTATTTCAGAGGAGTTACCCCATAAAACGACGCCATAGGAAAGAATGGAGTATGCATAAGCATAGTATGCTGAGAGAGCTGTTTGAAAGTTTGTA
ACGCGTTTCAAGTGGTGTAGTGCATATATGAAAGATGATAATTTATGTTCTACTTTTTTAATATGCTGCTTCCAGTTTAAATGTGAATCTAGTTCAATGCCCAACAGGGTTGCTGTGTTCACTGTTTCTAATTTTATATTTTTATAACAATATTGTACCTGTAATGAACTTTTTTGATAGGGCTTAAATTGGATTAATTTGGTTTTTTTAAGGTTTAGTTGGAGATTATGTGTTTCTAGCCAGTTCGTAATGTTGTCTAAAATAAAGT
TTAATTTAGTTTTAAGTTCCGATGAGTTTGAGCAAGAGATAAGCACTGAGATGTCATCAGCAAACAATACACACAGATTATCAAAAATATCAGGTAGATTATTTATATAAATGAGGAATAAGACACATCCTAGAACGCTACCTTGAGGTATTGATCCGGTAACTTGGATGGCATTAGATCTGGTGTATGTGATCATTCCTGTCTCAAAGTCTGTATTTTGTATTTCAACGTATTTGGAATTTGGAATTATTTAGTAGTATTTGGAAGT
ATTTGGAATTGTTGAAACCAATAAATAAAACTTATAAAATCCTTATTCTACTTTCTACCATTTCGTGAAACAAACAGTAGATGGCCGATGTTTTTGTGTTCTAGGCCCACTTACAGGAAAATTAATGGAGATTACATAGTAGATATTATATCTAGACTAACCCCCTTACTAATAAAATAATAAAAATAACCGACATCCACGTGGGCGGAGCCACGGGCGACCGCAAGTTTCTTATAAATCTGTTGAAAATTTACGTCAAATGTGAAAA
TTCATGGTCTTTCTTTTATTTCTGATATTATGATGTTATTTAGTAAAGAATAATCCTCACGATGAAACCTTTCGATCGGTATGATAAAGCTACAGGTTAATTATTTTTCTCGTGTAACGGGTTCGATTCCCGGTTCAACAATGTAATTATTTAAATATCTATGAATGCAGTTTAAATAGTTTCTGAAATTGAAATAAAGTCTTCTTTCAGTAAAACGTTCTAACTGTAATATTTAAGGTACTTTCTGTGGTACTTTCCCTTCATGTGG
CATAGCCGTGGGACGGCCTGTATATTGCTGCGTAAATAAATATCTTGCTGCCCAATTAATTATTGTGCTGATCAAATAATAATGTTGCTGCCCAAATAATTATTATGCCGCCCAAATAATTATAATGCTGACAAAGTATTTGTTTGCTGCACAAATTATTTTTCGGGTTACCTTTTTGTTGCTGACCAAATAATTAAGAGACTGCCTAATTAATTACTTCCCGTTTAAAAAACCTCGGAGTAAACAGAACAAGTCCTACAAAATTGTA
TTGAACGTTCATTTTCGATTCACATTAAAGCAAAGGGGCGTTCCACTAAAAATAAAAGTGTCTACGTGATTAACTAGATGGCGCTGTCGTACACCTAAATCGTGCTATAATTTTCTCTCCTATTCTATACAATTTTCAGTTGTTGTATCATGTAAATACCCTGGTCATAAAGTATATTGAAAAAGACTATTTGTTTTATAATTTTTCACTAGATCCTCAATAAAACACCAATGGGTTGTTTAATATTAAAAATATTTACTAATAGTTG
TGAACATAAGAGCTTCCATTCCAGATTAACTACAAACATACTCTTGCAAATATGGGATCCTGGTAGCTCTCCGCAGTTTGTCAATGTGACAAAATGAGGACACTAGTTTGTAGTTTCATTGTTTCCTAGTTTATTAGGCCACGCCTAATCGTATGCCATGCCACTCCACGTACGCAGTCATTGATAACCGTCTGGTTTCGATAATGAAAACAAGGATACTATTATTTTGGATGGACCTCGGATGGATGACCAAAACTTTGTGATTTCG
ACTTCCACTGTGCTTTGGAAGCTGTTGATCTCGGTGTGTCCTGTAGGATGTCTGCTTTTTAATGTGTAATGTGAAATATAGAAACTTCGTTAGAAAACTACACAATAGAATATTGTTGACAAGAGGCTATAGAAACCTTTAAAAAGTCAAATTATTTTTAAGATAACCATAATAAGACTTTAAAAAGTTTGTACATATACGTTTCTATTCACCTGTGTCTACCCCTTCGGCGATAGAGGCGTGATTTTTATATGTTATGTTTATATTT
ATCCTTTCTATTCTACATCTCCGTATCTTCTAAAAATGAAAAGTAATTTGAATATTGAATTTTCTGATCATTGATATGAGTAAGGAGGCTTAAGGGTTTGCATAAAAAAATAATATGAATAAAGTATTATCAAAGTAAATAAACCAAGAAGTAAATCTGGCTTTTCTCTATAATTCCTTAGCTCCGTTTGATTTTCCTATTAAAATAATTAGCAGTGTAAATTGAAAGAAACTCGATAATTACATGCTACTGACAGGGTGGGAGATCG
TTAATCAAATCGCAAGAGAGGGAGCCGGAAGACGTATTTCTCTCGCTTCCACTCATCACTACCTCCATTAACTTTCTCTTTCACTCACAAAATTCGTCGCATGCAATAACAGATTCACATTTTTCACAAATCTCTAAGTTTTAGAGTTTTTTTACTTTAAATTTTCCACGTCTAGTTTTAAAGTGTGGTTTTGTATAACATGAAATAAATATGAAAGCACTAGAGATAGCCTTATGAAAAGAATATATTCGTTGCTGCAGTGAAAATT
GAACCATAACGTAATGGCTGTACATTGTATTTCGTTTGCGTGATAGGCGCAGTGTGCTTCGTAGTTAACTCGTGATGTTGCAGTGCAGTGTTCTATTCTATTGTGGTAAGGGTGCAATTTGCTTGTTCCTGCAATATGCGTGTCTTCTTGTGGGTTATAGGAGCCTGGTAGGTGATGCCCTTTTACACGTGACGTCACCCCGAAATCCCCATCCGCTGTCCCGTTTTCTCTTAATGGAGGGTGCTTTTTCTTTCAAACAATGCAATGT
GAGGAAAGCAGTTTTATGCCGCTTTTGGATTACGGATTTCTAACTACTTACTAACATATTGGGTCCTTACCTATGAAATTGGTGTTTTTTTTCTCTCTTAGTATTTTGTCTGTAACTATTAAGATTCAGAGTATTCCTAAAAAATAATTACCTCGTCTAAAAATAGACTATATTTTTGAGCTCTTTTGATTTGTTTTGATAATTTGTTGTAAGTTAATTGTGTTAAAATTCGACACGGAGACAACTCTTTTTAAGTGCCTCCCGAATA
CATCATTTTATTTGAATTCACTTAAAAACGATAAATAATTTTTATGAAGTGTGATATTACACATATTACTAGAGCTTGTCTAATGATACATGCCAAATATTATTATCTTTGGATTTGTCGTTGTGAAGAAAACTGAAAAAACTTTAGCATGGAAAATGCAGAATTCCCCAAAATCTCCATACAAAACACCAATTTCATAGGTAAGGACCTAATATTAATACCGCATATTAATTACTAACACCCTTACTAATAAACAAGGTATAAGGTT
GAAATAGTTATTCAGTGTTTTGTCTTCGTCAGACATTCAGAATACAGTGGTTGTCAGAAAGTGACAAAACACTGAATAACTATTCCTTCCTTATAACTCGTTTATTAGTAACGGGGTTAGAATATTTAGAATGTGTCCCAATACCTAACTTTTCATCACACAAACATTTGCCTCGACTTGGCTTTGAACCCAGAATCGCTAGAATCGCTTCCATTCTTTCACGGAGGTCGATTCCCAATAAACTCAAAACCGAACGTTGAGATTACCA
TTTTCAAAGAATGGAAGGGTTTTTGTTGATAAAGTTGAAGGTTTTCATTCATTTTTTAAATAAACGTTTATTTTCAATAAAACTAACGCATAAATAAGTGGTCATGAAGTAGAACATTTTGCGGCACAGCTTCAGTACTACGAATTATGCATGTTCTGAAAACATCTTATACAATAGGTTTTTTTTGTAATAGAAGAATGTCTATACTCACTGCCAAAAAAACAACACATGATAAGTATTACTAGTACTAGACCAATGAAAATAAATT
CCCCAATATGGAAATATGTTTCCATATTTTGAAGTGGCTCTCCGAGATTCCTTTGTGTCGTCAATAGAGTGCTCATGTTAGATTACTGAATGGTCTGTAATAACGCGTATAGAAAAGTTTTGTTTGTTGTTCCAGGATGGGCTAGCGCCCCACTCTCACTATATGGTTATGATAAGCTATATGTATGTTTACGATATGTAGTTATTTATACGTCGGCCGTAACGTTACTAGCCAGATCTTTGTGCATGTTATGTTGTTGATTGTGACT
GAGTAGGTTAAAAAAATTTTTGTTATGGTTTTTCAAAATTTCTTATAGCAGAATGTCAAATACACTATTGGATGCTACTATGGCTCTACCTTTAACTATTTTGTATGTATACATATGTATTCATATTTCCTATTATAATTATATTTCTTTTCTTGTTTTTTTACATAATTTTTACGTTGTCAATTATAGCTGTTAGGTATCGGAGATAAATAAGAAGCCGAGCTTCCGTCTAACAGTTTTTAATAATTAGTAATAAAAAATCAATGTC
AAAAATGGCAAGAAAAGAATATAAGTCAAAAATTAAAGAAAACATAAATGTATCAAAAAGTATGCTACCTACAATGTGTCTATGGCCAGATCTGTAGCCAACAGAACTACCCCCCAGTGAACGCACGAATTCAGGGTGGCGTACAGGTACTGGCCAAGGACACCAGAAGCCGGGTCGCCCGCTTAAACGACCCCGCTCCAGTGGCAACATCGACGACTCTAACTATGCCATCTCGCCCGGGGTACAGTTTTGTGATCCGGCCGCGAGG
CCACGAACCACGGGGGAGGGTCCCATCAACGATTAGAACCAGATCTCCAACTTTAAAGTGCCGGCTTTGCCCAGGCTTCCGCGGGTAGAGTGTAGGCAAGAACTCCTTAGTCCACCTCTGCCAAAAGTGGTCGGCGAGACGGAGGCCCTTTTTGAAGGATAGCCGACCAAATAAATCAGAGTCAGAGAAAGTTGAGAGAGGTAAATCATTGACGGGACCCAAGAGAATGAAATGAAAAGGCGTGAGCGCCTCCGGCTCTCCTGGCTCC
ACGGAAACATGAGTTAGTGGCCGGCTGTTCACAATGTTTTCCACCTCCAGAAGCAGCGTGTGCAGGACCTCTTCTCTGGGAGAGCGCTCCTTCAGGGTCACAGCCAACGAGGTCTTGACGGTTCTCACAAGCCGCTCCCAGGATCCTCCCATAAATGGGGCTGCAGGAGGGATGAAGACCCACCTGATTTTCCTGTCAGCGCTGAACTCGTAAATAGCAGGTAACAAGCGCGCAGCGCCGACGAAAGCTGTTCCATTGTCTGAGTAGA
TGGACTCCGGGCACCCTCGACGAGCGATAAACCTGCGAATCGCCATAATCGCAGAGTCTGAGGAAAGCGACTCTACCACTTCGAGATGTACTGCTCGGACTGTCAGGCAGGTAAACAACGCTACATAGCGTTTCAAATGGCGCCTTCCTTGCGCAATCGTGACTGGCCCGAAGTAGTCCACTCCAACATGAGAAAACGGCCTCGATTTATAAGCTAGGCGTGCCGGTGGTAGGTTTCCGAAGGGTGGTGTCACTGCTCGGGCTTTCCG
AATACGGCAAAGATTGCATCTGGATAAAACAAGCTTCACCGATGGTCGCAGCCGAAGAATGAAAAACCTTACTCGTAGCGCGTTGACCACAGTCTCAACTCCGCTATGCGCCATCTTTCGGTGGTAGTCTCCGATCAGCAATTGCACTATTGAATGTCGACCGTCCAAGACAATTGGCTCCTGCTCATGGCCTGGTGGCAAAACTTCTGTTAGGGAGACCCTGATAGAGATCCGGAGGAGATGATCAGACCTAACAATGAGTGATACA
TTTTTAAGCCTACTGTACCTGGGGAGTGGCATAGAATTTCTAACGCATGTCATTTCCTCTGCCAAACTATCAACTTGAGACTTAAGCAACAATTGGCTTTCGGCTCTAATTACATTATCTGCCGAAAGTAAACCCGGCATTTGGGATGGACGCTTTAAGAACACGTTCGCGCAAAAATAAATACGGGCAGTTGCACGTAAAAGGCGTGTCCACGAGCTGAATTTCGTAAAATCTGCAACTACGGGTACAATAGAAATTAGTACTTGAC
TTTCAGACGAACAATGAACCATTTCAGGCAAAGGAAATGACACTTGGGGTTCCTTGGGCCAATCAGAATTAGAAGCCAGGAATGATGGCCCTAAGAACCAGCGGGAAATTTTGAATTTGTTGTCTGGTCTCGTAGCGTCGTCCGCCACATTTTGTGCTGAAGTCACATAATGCCAATCCGAGACATTAGTTATTTCAGTAATTTCCCCAACTCTAAGAGCCACAAAGGCTTTCAGTGAGCGGGCGTCGCTACGTATCCATCTTAAAAC
GGTCAAACTATCAGTCCAAAAGTAAATTTTGAAGGTTTTCGTCGGTGACAGTTTAGAATGTAGGCACCGAGACGAGCAGCGATTAACGCAGCTTGCAATTCTAACCGCGGAATTGTCGTTAGTTTGAGCGGTGCTGAGCGAGTCTTTCCGGCGATCAACGATATCTTAAAAGTCTGATCTGAATACACAAAACGCCAGTATGCCACACACGAATAGGCAAGCTCGCTTGCATCCGCAAAAACGTGGAGCTCAACCTCTGAAAAGGCAG
AGTCGCTGAAATAGCAGCGAGGTATATTGACATGTGCAACATCTTCAAGGTCTTTAAACCAAACGTACCACAGGGCTGATAGACTTGAAGGCAGCTCCGCGTCCCAATCAGAGACGGCCTTCCAAGTCTTTTGAAAAAGTATTTTACCCTTGACTGTAATAGGACTTAGGAGACCTAGAGGATCATAGACGCTCATCAACCTTGACAATACATATCTCTTTGTTAGGATGCGAGGGACAGTTACAGTTAAATTTCGCGTAGGGTGAAT
AGTATCAAGTGTTGTATTCCAAGAGACTCCTAAAACCTTAACGCTGTCATCTGAACGCGTGGCACGCAACTCTCCCGGAACCAATGACAAAGCGCCCTCTACGTTAGAAACCCAGGACCTCATCTCAAAACCCGCACGAGAATGAACGTCAACGACATCCCTAGCCAGATGAGCCGCCTCAGAAACATCATTAACCGACGTTACGAGATCATCCATATAATGGTCCTGAATGATTACATTAGCGGCTTTCGGAAACCTGTCCTTATGC
TGCTCAGCATTCAGGTTCATTAGATAGAGAGCCGTGAAAGGACTCGAGGTAGCGCCAAAAATCATGGACGACATTCGGTAATGACATAAGGGCTGAGACGGATCAGCGCGCCACAGAAACCGCTGAGCGTCACGATCTAACATGCGGATTTTTATCTGAGGGTACATCTCCCTAATATCGGCGTTTAGGGCTATCAGTCCTTCTCGAAAACGCATAAGGACCTCAAGAAGGGGTTGAAGTAGGTCGGGCCCTGACAACAAAAGGGAGT
TTAAGCTTACTCCTTGAGCCTTTGCTGCGCAATCGTGAACAACCCTAAGCTTTTTCTTCTGTCTATGGACAACACCAAAATGTGGCAAATACCATTTTACCGGAGTATCAGCGTGTGGCATCTCGCACTTCTCAGCATAACCCTTGTCTATCATGGCTTGAATATTACTACGATACTGAGTTGCATACTCTGGGTCCTTAGCCATCTTTTGTTCTAGAGAAGTAAATCTGCGACGTGCTAGAGCGTAACTATTAGGAAGCTGTCCCAA
ATTAACATCTGACCGCCATAGTAGCCCTGTCTCAAATCGACCAGAAGGGAGTCTGCGTGTGTTGACACGAAGGGTTTCTATGGCGAAAATATCATCCTTGTCAAGTCGCTCTCTCCTATCAATACCAATTGACTCTAGTTTAAAATTTTCTCGAATAAGATTCTCTAGAGAATTATCTCGTACGTGATTGACAGTAGAGTCTGACCGGAAAACTCTACTACAGACGTTCTACTTGTCGGGAAACCGTAAACTACCCATCCTAAAGGAG
TACGACATGCCACAGGCTGAGCGCGTGAGATGCGCCTAACTTCCGACGCTACCAGCAGGTGCCAATTGTCCATGCCGACTAGAATTGATGGTTCCACGTCCGAGTAACTGCACAAATTAAGACCGTTAAAATGCTCCAAATTAGTATCGCATCTCGATAAAGACTGACGAGAGAGCCCAAGATCGCGAACGGAACGCACATGTTTCAAAACATGTGGTTGACAAGTCTTATCATGACCTTTAATTGTAAAGCTAACATACTCCACGTC
TACTCGCTTTTCTAGATTACGCACACATGAGATGATAACACTCTTTTTTAGCCCCGTAACACCGATAGCAGACGAAACCTTAGCGTCTATGAACGTAGCAGTTGAACCCTCATCCAGAAGGGCAAAGGAATCTATGCTGCCACTCGGACCACTGACGGTTACCGGCAAAATCTTAAGAAGTGGTCTGGAATCTTGCTGCTTACATTGATCGCCATCTGTTTCCAAAACTGTCTGCCCCAACGAGTAAACCTGGGTTGAGGAGTCGACT
TTAGACCTATCGTGCAACGCATCGAGTTCGGTAGTCGGTTCTGCAGAAAATTGAGAACCTACAACAGGATCGACATTGGTAGCTGCTTCATGCAAGAGAGAATGGTGGTGACGTTTACGTCGGCAACCGACGACCTTACAACATTTCTTGCAGTTTCTATAGCTGTGACCACCTTTCCTCAAGCATTTGAAGCAAATAAAATTGTCTCTTACCCAACTCAATCGTTTTTCTGCAACCAAATCACGAAACATCTGACACTTCTGGATTT
CATGCGTAGCCTTACAATAAGCACAACCAGGTTCGCTAGAGGTGTTATCTGTGTGACTTGGAAACGATGACTTAGGAACTTTACCTGGTTGTTCGAAGCCTTCTTTGTGTCTCTTGACAGAGCGAAGATGTCCATAATTAGAGACAGGCCTTCCAGTCAAAGTCGTTTGACAGACTGTAGAGGGGTGCTGGTCAAGAGTTGCTGAAGTGAGGGAGGAGTCGACGAAGTCAGCCTGGACAAAATGAGCATGAGACCTATCTGACTTAGG
TTTAAAATCTGTCAGGATACCATATCTTAAGTGCAATAAAGCCTCATGCGATAAGAAATCAGAAAGCTTTTCCAAGCGAAACTTATTTTCATGCTCTTTCTGAAAGGCAAACGCCAACCACTTCTCCCTCAGGAGAGGAGTAAGCTTTCCAAGATAACGTTGCACAGTTCAGGGTTAGCTAAATGGACTTGCTGCTGCAGAAGCTGAACGGTACTTATGCAGTTGCGTACCTTGCATGCAAAAACATTTAGCTCCTGTGCATCGTACC
CGCTTAACAGCGTAGCCGCTTCTGCGCAAGAACGGAAGAACATTTGGTGGCGACTGAATTACCGCTTGAGGAACTCACCTGCGCCCTTTGCTTCGAACGTGACGTGGATGGGGACGGCGGCGGGCCCAACTCTGCGACCGCCGCGGCGGCATTATCCGTAGCGGAGTTAGCCTGCTGCTTTGCGATAGCACGGGTGATCATGCCGCGAACTTTTAAATAACTTCTGACACCAGAAACTGTTAGGTATCGGAGATAAATAAGAAGCCGA
GCTTCCGTCTAACAGTTTTTAATAATTAGTAATAAAAAATCAATGTCAAAAATGGCAAGAAAAGAATATAAGTCAAAAATTAAAGAAAACATAAATGTATCAAAAAGTATGCTACCTACAATGTGTCTATGGCCAGATCTGTAGCCAACAATAGCTATTAATTTTTTTGTTGAATAAAATACTACACATCTACTCTTACCGCGAATCGGACCCTCAGATATGGTCACGAAGGTCGTAAATATTTTTAAGACGTTGTTTCAAACCATCG
CGTTTGAATATAACGTATCATCGTTTGACACCCTTCGGAGAAAAATATTTGCCCAGCAGTGGGAGTCTTTCGACTGAGATGTATACCATGGCTGAAAAAAAGATACTGAATAAGAACACTCGAAAAAAAAAATCGGCATTCGGCGATCTTAACGCTATGCAGCAGTCTCTTTCAGAAAAACTGCATCGTAATAATACTCGTATAATAGAAAAAATCAATCGTTACACAGTTATTTTCAAGAACATTTGTATCATATATTTGTAAATAT
TCTTTTTTATATTGCAAGATCATTTCTTAGTTATTGTTAAATGGTTGTTCCTTTGCGACCCATTCAATGTCTGCGGTCACCAGTCGACCGTTGTTGACAGCGTGTCAGACTGAGAAGGGTCTATCGTTGGAATAACTGACATATTAACACAGTACTTGTACACATGGAAACGATTTTATATAATACGACTAGAATTCTATTGTGATAGTTTTTTTGTTGATGCTAATGAATACCTGGCAGTATCTATAGACGTAGTTACAATTAACCG
TATTTTAACAGAATTAAATTCAAATAAATAAGTGTGCCTATTAGTATTTTTATTAGTTACTAGTGGCTTACCGCGGTTTCACCCGCGCGGTACCCGTTCCAATGGGAATATCGGGATAAAAAGTAGCCTGTATTCTGTATACCAAATTTCAATTAAATTGGTTCATAGTTTTGACGTGAAAGCTAGACTGACAGATAGAGAAATATACAGACAGAGTTACTTTCGCATTTATTATTAAGTATGGATTGTTATTATTTTATTTTAATTT
ATTTACTTGGAAGAATCGTTCACTCAGGTTGAACGTAGAACCCTAATGGACCATCGATTTGTAAAATAATAATAATAATAATATCCTCAGACATTCACACCGATCAACTAGCCCCATTTTAAGCAACTAATGCTGCTTGTGTTACGGACACTAGATGACGGATAAATATATTTAATAGATAATAATAATAATAATAATAATAATAATTTATTTCGATAACTTAGTCTACAATTACAAATTAAATGTGGGATCTCCCTTTTAGGCAAGA
CATCGCCTATAGATAAATACATATAAATAACCAGAATATATAGAAATATATATAAACATCCAAGACTGGAGAACAAATGCCTGAAAATTCATCACCCAAACATTTGCCCCCACTGGGGATCGAACCCAGAACCTCTCGAGATGGCGACGCCTTGAGACCGGCATACAGACCACTCGACCACGGAGGTCGTCAAAAGAACGAGGAAATGCGTTGTATGCGGGCTGCACAGGACCCACCTGCTTAGAGAATTGGAGATCCTTATGGGAGG
CTTGTATTGTATCCAACAGTAGGTGTCTGTCACCTGATATGACATATTGGCGAGTTTAGGTCATTACCTTGGTTTTACATACAAATAACGTATACTCTAAGGTTCCTGAATCTATATAATTTAATTATGAAAATTATCGCATTCGTCTCTGGAATAACCTTCCATTACCAATCAAACAATTCCAATCCTTAGCATCCTTTAATGATCTGGTCCATACCCATTTCCTTCATCTTTGACTCCTTCCTCCAACACACATTGCAGTTTTCGC
CTTTTCTTTGTTGCTTTTTGTGTAAGAATATAGATAATAAGAATATATTGCTAAATAAAATATTTATACAGGATATTGTAGCATTTATAATATAGTATGTATTTTATGTATGTATATAATATATTATGTAATTTATATGTAGTAGTTTATATTTGCACCTTATATATGTTACCTACCTTTTCTTTCATTCTATCTCTGTCCAAAGGTTGTCTGGAAGATATTGCTATTAGCTATAAGGCCGCCTGTGTTGTATTGTATTTTTTTACTA
TATTTCTGTTTTAATTAGTTGCAATAAAGAATTTTATCTATCTATCTATTGCCGATAACTTTCATGAAGAATTTTCCTATTCATGAATACCATGAACAACAAACCTAAACTCACCCTTCTCACTCAAGTTAACTTATGCCAACTATAAATAATAAAAATAAACGAAACCCACATTATGATTGTGTAGTAAAATCTGACAGTAAATTAATATTTCTATTTGGTAAGGCCGGTACATTTAAAGCGTTTTGTTTTAAAAATACAGCTGGCT
GTGTGCCAAGCAAGCGAATACTATATTGGGCCAGCCTGGCTAATTGTTACTTCTGAATGCAACGAGGGAAATTTCTACAGCCTCCGACAACATTGTGGATAATTTTTCGACTGTATCCAACGTTGTAGTTCCTTTACAAGAATACAAAAAGCTCTTTAAACCTATTATGAGTCCTGTGCGCATTTCGGAATTTCGAGTTGCGTTCGAGTTTTTCACTTGTTTTACCAGGAAGTTATTGTAGGGATGACTGTAATTATGATACAAACAT
TTTTGATGATGTTGTAAAAAAAAAGCTATTGCATAGCATTTTGTATCAACTTATGCAATTATAATTTTATTTATTTATTTTATTCATGCAGTATTTCTTTTTCTTTTTTATTATTTTACAATACATGTGGGTTTTATTATCTTAACGCCGTGTCGAAGACTGCCGAACTGAAACGACGTTAGACGATGCACGGCCTTCAAGCCACACCTCCTTGCGCATCGGAGTGGGGAGCGTGAGGTTTTTTCGTTACGGAATTTCTGGATTCGGT
CCCCGCGCTCAAGGCCCGAGATAGAAGCTATGCAATAGCTTAAAAGCTTGTAGAGAAAGAGATAAAACGCCTAAAAATGGAGACCTAACATATCAGCAAAATATGGGTGATAACACCTCCCTACCCCTTTGCAGAAAGAGGCGCGATGATATGTTATTCCATATTTTTTTTACATATTTACATGCAGTGTTATTGTAAATGGACAACCTCCGTAGCCGAAATTTGTACGCTGGTTCGAAGGAATCTCGTCATTTTTAGAGGTAGGCTC
ATTTGATCTTCGGTGATTTATTCTCTAAAACTTATATGTATTGAATATTATGGGCCCTGGCAGGGAAAGTACCTTACATATTTAAAATAGATAAATTTACATAAAGGACTAACTTTTGTTTTTAATAATTAACATAACTGATCTCTCAGTTTTTTTTTAAAAAAGCTTACCCAAACCTTCCCTGGAATATAAAATAATTGTTAGACATGTTTAAAACGTAAAGCTTAATAAAGAAATTAAATTTTCAACAATGACTTAATCGGTTAGT
GGAATTAAATCGCGAACTTTTTTTATTCATTTTTTCCTTATTCGATTCTCGGTTTGAGTAAGCTAGCTAGCTATTTTGAGAATATTTAGAACTTAGTTATGTTTCTTATTCAAAGTAGAAGTATGCTCTTGTGTAAATTATATTTTTTTTTAATATTAGCTAATAGCTCCAGGGCCCACAAAATTTTCCACTCTGTATGTAGGTATATACTTATCTATTACGCTCATTAAAACTTATCCGTCATTTAGTATCCATAACACAAGCAGTA
TAAGCTGCTTATTTTAGGCTACTTGATAGGTGAAAAAAATATGTATTACATTCATACATACATTCACGCCTCTTTCCCAGGGGGGTTGGCAGAGATTACAGATCTGCACTTGCTACGATCCTGCATCCCGCACATGACACACACTGTCTCTACCCTCATTACTAACTTTATACATGCCCTTCGGTTAATTTTAATAATTATTTATTAGAATATTAAGCTTTATTATGATTAATAGCGATGGCAATTCGTTAGCTTATAGGCGCCATTT
TCTTTATCCTCTACTTAAGTAGTAGCCAGCAATTTTAGGGCAGTGACCTAAGGTCAACGACTTGTAAAGGATAGTTCTTATGTTTTAAGAATACCGCATAGTAGATACGAATAACGTTGCAGTCTAACCGTGCAGAAGATTTTTGTCTTTCCGGATGATTAGATGTTATGAATATGAGGGAAAATATGCTTTAAGTATGAAGACTATAAGGTCTAGTATTTAATTTGATGTTATAGTTATCTTCGCACTGCTCTATAGATTCTTTTAT
ATAAAAATGACTTTTATTTTTTTATTAACAGCAACATTCTAATCTAAAATAATATAGACCAATTTTATATATTTACTTTTATATTTACCTGTTAGATTGAGACTGAGAAATATCAGTCGAATGGGAGTGTTTAAAACGCCTGTCTACCCCTTTGGGATAGAGGCGTACGTTTATGGTTTATTTTGATGTTTGTTGTATGACATATTACACTAAATCTCTACAAAGAAGAACAAGACGAGCCGAGTTGCCATGCATCGGGCACAAAGTG
AAGCAAGCAATATTAACAATGAACTGTACATAACTTTTAATTCAAACTCGGTAGAACTCGCGAATCCCGCGGCCTCTCCAAGGAGTAGTGCCCTCTGTCCCAATTCAGCTAAGGAATAGTCAACGTAATTACTAAAGCAGACTTATTGTCTCCACCACATGTAAACCGTTTGAATCATGCATTAATAAAACTTCATGTCCCCAACAGTGGGCACTGGCTTTTACCTGACTTCATATCTTACACATTTATATCCTTAGAGGAGTATACC
GAGGGGTCATGATTTTTGTAATCTAACAATGTATGTTTGTATTTTAAATTGATAATCCAGACTTATTATAAATGCGAAAGTAACTCTGTCTGTCTGTCTCGCTTTCACGTCAAAACTACTGAACCAATTTAACCGAAATTTAGTACACAGATAGTCTAGAACCTGAGAAAGGACATAGGCTACCATTTATCCCGATATTACCATGGGAACGGGTGCCATAGGGGTAAATGTAAAAACATTTAAATAACGCCTCCCAGAAAAATGAAGG
ACAAATTTACGCCACTATGAACTTTGCTTCTAATAATAGAGTAGGAGTAAATTTTCAATTCCACCAATTGAGGCTACGGTTCTTATTTTAAAAAATCTTTCAGTGTTCAATAGCCTATTTTTTGAGGAAGGCTATAGGCTACATACCATTAATCACACTATCTCAAAGTCACTATTCTACGCGGATGAAGTTGCGGGCAAAAGCTAGTTGATAAATTCATTGATGTATTGATAATTCATAGACCATCAAGTCATTTAAAAACGTAATA
AAAAAAAATGTGTTAAAAGCCTCAAAAAAAATTATATAATTTTTTATGCTGGTGGCTAACAGAAATGAGTAATTTGGGGCCCAATAAAAAAAGGGTCAACTAGCCTAATATTTTAATTTCTTTTTTGTACTCATTATATTAGGTCCTTTCCTATGAAATTGGTGTTTTGTATGGAGATTTTGGGGATGTCTGCATTTTAAATACTAACATTTTATCAGTTTTCTTCACAACGACTAATCCAGAGATAATAATATTTGGCATGTATTAT
AAGACAAGCTTTAGCAATATGTGTGTGAACACGCTTGATAAAAATTATTCATCGTTTTTATGTTAATGCAAATCAAATGATGTATTCGGGAGGCAGTTAACGTTTAAACATCAGCCTATGTTTGTCCACTGCTGGACATAGGCTTACCCTAATGCATCGAGAGGTTATTCGAAAGATTAATTGCTGTTTAGCTGAAAGGTAAATTGTGAAATCAATCTAGTTTCAAGAAATTAACTGTTTATCCTATGTGGAGTTATCAGTAAAATGT
TTTTCCACTAAATTGTTAGTTAAAATTTTACACAGCAATCCAACATAACTCTTCTAGTCATAGCAGCAGAGTGGTTTTTTAAAAACCGACCGTTTGTTGCCGACACCACACCTGTTTCGCAATTTTTACAACATTCAAAACACCACAAACTCATTTAAACTACACTACATCAAATTGTCACGTTAAATTTATTACAATAGAGTTGCTTTTTGTTACAAAAACCTTTAAAAATGTATAGTTTTACGATATTGAACTTTAAAGCGGTAAT
AATAAAGCCTTAATATTGTGTTTTAAGACTATTATCTAACGACGCCGTTCGCGTAATTTTAGTTACATTGATCCAATTAATATGAAAACTTATAGCTGCTATCGTGTTTCGACACTAGATTTTCTTTGACACCAAAAAAGTACTAAGCTTATTACATAAAAGGCCAAATAGTGTTATAATTGTTTGTTTTGACAATCACTGATGAAACTTTGATATATTTTTCCCGATGTAGGTATTCGTCTTAGTACACTACTTAAATAAATAGAAC
ATTTTACTAAAATGTTCTTCATTGTAGTAATTATCGCTATTTCTTTTGAATCAAAAATTTAATAATATTAACGCGACGTTTCCCGATCACGCTATATTCCCGAATGGGTAGTTTTAATCTGAGCAACTTTACAATAATAACTTTTTAAATCATAGTTGATTAATAAATAAATTATTCAAAAAAAAATACAAATTTAAGACTAAAAATACTTAAGACAAAATTATACTTACAGACTTTTTATTATCATTT
CIRCULAR CONSENSUS SEQUENCING (CCS) MODE:20 KB HUMAN HIFI LIBRARY – READ LENGTH AND RAW BASE YIELD
PERFORMANCE
Data shown above from a 20 kb size-selected human library using the SMRTbell Template Prep Kit on a Sequel II System (2.0 Chemistry, Sequel II
System Software v8.0, 30-hour movie). Read lengths, reads/data per SMRT Cell 8M and other sequencing performance results vary based on sample
quality/type and insert size.
METRICS
Insert Size 20 kb
Number of Raw Bases 377 Gb
Total Reads 4,266,403
Half of Bases in Reads (bp) >193,403
Longest Read Lengths (bp) >300,000
Customer Average:
325 Gb Raw Bases
METRICS
Insert Size 20 kb
Number of HiFi (>Q20) Bases 26 Gb
Number of HiFi (>Q20) Reads 1,423,277
Mean HiFi Read Accuracy 99.92%
CIRCULAR CONSENSUS SEQUENCING (CCS) MODE (CONT.):20 KB HUMAN HIFI LIBRARY – SINGLE-MOLECULE ACCURACY AND
HIFI BASE YIELD PERFORMANCE EXAMPLE (POST-CCS PROCESSING)
Data shown above from a 20 kb size-selected human library using the SMRTbell Template Prep Kit on a Sequel II System (2.0 Chemistry, Sequel II
System Software v8.0, 30-hour movie). Read lengths, reads/data per SMRT Cell 8M and other sequencing performance results vary based on sample
quality/type and insert size.
≥Q20 (99%)
HiFi accuracy
WHOLE GENOME SEQUENCING APPLICATIONS POWERED BY HIFI
READS
De novo assembly of genomes with high
contiguity, correctness, and
completeness
1 Human Genome
per 2 to 3 SMRT Cells 8M(~70 – 100 samples / yr / Sequel II)
High precision and recall for single-
nucleotide variants, indels, and structural
and copy number variants with HiFi reads
1 Human Genome
per 2 SMRT Cells 8M(~100 samples / yr / Sequel II)
- For researchers who want to detect variants comprehensively in a
whole human genome, the Sequel II System provides high precision
and recall for single-nucleotide variants, indels, structural variants,
and copy-number variants, including in repetitive regions of the
genome.
- HiFi reads combine the benefits of high accuracy and long read
length.
- Recommend using 2 SMRT Cells 8M to achieve ~15-fold HiFi read
coverage of a whole human genome for comprehensive variant
detection
- Variant calling with HiFi reads can be performed with standard
software tools like Google DeepVariant and GATK.
- Run ~100 human samples (3 Gb) per year, per Sequel II System*.
VARIANT DETECTION APPLICATIONS
POWERED BY HIFI READS
Gain complete views of human genetic
diversity with PacBio long-read sequencing
* Read lengths, reads/data per SMRT Cell 8M, and other sequencing performance results vary based on sample quality/type and insert size. Prices, listed in USD, are
approximate and may vary by region. Pricing includes library and sequencing reagents run on a Sequel II System and does not include instrument amortization or
other reagents.
HIFI READS ENABLE HIGH PRECISION & RECALL FOR DETECTING
ALL VARIANT TYPES
Variant calls from ~15-fold HiFi read coverage of a human genome (HG002) were measured against the Genome in a Bottle small variant benchmark
(v3.3.2) for SNVs and indels using Deep Variant and SMRT Link 8.0 for SVs. Libraries were generated using a 15 kb insert and sequenced using
Chemistry 2.0.
Perc
en
tag
e (
%)
99.1
99.6
95.0
96.6
97.7
95.2
90.0
91.0
92.0
93.0
94.0
95.0
96.0
97.0
98.0
99.0
100.0
Recall Precision Recall Precision
SNVs Small Indels SVs
Recall Precision
15-FOLD HIFI READ COVERAGE RECOMMENDATION FOR
COMPREHENSIVE VARIANT DETECTION APPLICATIONS
15-fold HiFi (≥Q20) Coverage[2 SMRT Cells 8M for a 3 Gb genome] provides a good trade-off between cost and results
Wenger, A. et al., Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome. 2019. Nature Biotechnology.
- HiFi reads provide assemblies with high contiguity, high base
pair accuracy, and high gene completeness.
- With megabase size contig N50s, accuracies >99.99%, and phased
haplotypes, you can do more biology – capturing undetected SNVs,
fully intact genes, and regulatory elements embedded in complex
regions.
- Compared to using standard PacBio Long Reads (via CLR
sequencing mode), de novo assembly using HiFi reads
generated with the Sequel II System affords smaller file sizes
and faster analysis times.
- Assemble up to a 2 Gb genome in a single SMRT Cell 8M for
~$1,300*.
- Run ~200 samples (2 Gb) per year, per Sequel II System*.
DE NOVO ASSEMBLY APPLICATIONS
POWERED BY HIFI READS
* Read lengths, reads/data per SMRT Cell 8M, and other sequencing performance results vary based on sample quality/type and insert size. Prices, listed in USD, are
approximate and may vary by region. Pricing includes library and sequencing reagents run on a Sequel II System and does not include instrument amortization or
other reagents.
PacBio is the only single-technology solution
for easy and affordable generation of high-
quality de novo assemblies.
GENERATE CONTIGUOUS DE NOVO ASSEMBLIES USING HIFI
READS
DATASET RICE DROSOPHILA HUMAN
HiFi Library Insert Size 17 kb 19 kb 15 kb
HiFi Read Coverage 20-fold 20-fold 22-fold
Contig N50 (Mb) 10.7 6.5 30.5
Obtain high contiguity assemblies with HiFi data; high
accuracy allows assembly through very similar repeats
GENERATE COMPLETE AND ACCURATE DE NOVO ASSEMBLIES
USING HIFI READS
Accuracies >Q40 (99.99%)
>98% of genes in frame
DATASET RICE DROSOPHILA HUMAN
Assembly size (Gb) 0.400 0.150 2.92
Base pair accuracy
(Phred/Percentage)Q50 / 99.999% Q50 / 99.999% Q49 / 99.9987%
BUSCO completeN=1,440
98.7%N=2,799
98.9%N=4,104
94.9%
Species-specific
genes in frame
N=19,313
98.7%N=19,947
98.9%N=35,666
99.5%
GENERATE DE NOVO ASSEMBLIES QUICKLY WITH HIFI READS
Compute times for de novo assembly of a human genome
DATA TYPE HIFI READS LONG READS
File Type CCS.FASTQ.GZ SUBREADS.BAM
File Size (GB) 48 323
Read Correction Method CCS Analysis Pre-assembly
Time to Results
(Hours)
Read
Correction17.5 43.5
Contig
Assembly13.7 18.9
Analyses run with PacBio recommended compute
infrastructure~31
hrs
~62
hrs
Assembly with HiFi Reads takes half the time of
assembly with Long Reads.
COMPARISON OF DE NOVO ASSEMBLY RESULTS FOR CALIFORNIA
REDWOOD TREE (ESTIMATED 27 GB HEXAPLOID GENOME)
[1] Hybrid assembly of redwood using ONT + short reads (2019).
[2] Douglas Fir assembly by Neale et al. using short reads (2017)
[3] Transcript set of Abies alba from Neale et al. (2019). Varying number of transcripts aligned to each genome (4,958
mapped to PacBio HiFi redwood, 4,760 mapped to ONT redwood, 16,187 mapped to Douglas fir)
PacBio’s HiFi de novo assembly of the large California redwood (Sequoia sempervirens)
achieves markedly improved performance in the 3 C’s of genome assembly quality (Contiguity,
Completeness, and Correctness) compared to previous conifer assemblies using other
technologies.
A 24Kb PacBio HiFi Library was prepared and sequenced and the raw HiFi data was accessioned and deposited in NCBI under BioProject PRJNA606797.
HIFI SMRTBELL EXPRESS 2.0 LIBRARY PREPARATION &
SEQUENCING WORKFLOW SUMMARY OVERVIEW
1. gDNA QC & Shearing
- CHEF Mapper, Femto Pulse or Pippin Pulse sizing QC
- Shearing with Megaruptor or g-Tubes
2. SMRTbell Express Template Prep Kit 2.0 Library Construction
- Perform single-tube, addition-only reactions
3. Size-select & Purify SMRTbell Library
- Perform size selection using SageELF for HiFi Variant Detection or BluePippin for HiFi de novo
Assembly applications
4. Sequencing Preparation (CCS Sequencing Mode)
- Perform Sample Annealing / Binding / Cleanup (A/B/C)
- Follow QRC for loading recommendations
5. Analyze
- For variant detection analysis, can use GATK or Google Deep Variant
- For large genome de novo assembly, can use Falcon-Unzip or other third-party software
Workflow summary for constructing SMRTbell libraries ~11 – 20 kb (or larger) that
are suitable for generating high-accuracy long reads on the Sequel II System using
SMRTbell Express TPK 2.0 for de novo assembly and variant detection applications
~4.5 hours
HiFi Library Sample Preparation Workflow
Details
PROCEDURE & CHECKLIST – PREPARING HIFI SMRTBELL
LIBRARIES USING SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0
- Procedure & Checklist describes a method for
constructing SMRTbell libraries ~11 – 20 kb (or larger)
that are suitable for generating high-accuracy long
reads on the Sequel II System using SMRTbell Express
TPK 2.0 for de novo assembly and variant detection
applications
- Protocol document contains:
1. Recommendations for gDNA QC and quantification
2. Recommendations for shearing gDNA to the desired
target mode size using either the Megaruptor System
(Diagenode) or g-Tubes (Sage Science)
3. Enzymatic steps for preparation of a HiFi SMRTbell
library using SMRTbell Express TPK 2.0
4. Instructions for size-selection of the HiFi SMRTbell library
using either the SageELF System (Sage Science) or
BluePippin System (Sage Science), and also includes
protocol reference for performing AMPure BP Size
Selection method for de novo assembly applications
using HiFi reads if required
5. Sample setup guidance for preparing HiFi libraries for
sequencing on the Sequel II System
https://www.pacb.com/support/documentation/
HIFI SMRTBELL EXPRESS 2.0 LIBRARY PREPARATION &
SEQUENCING DETAILED WORKFLOW OVERVIEW
1. gDNA QC & Shearing
- CHEF Mapper, Femto Pulse or Pippin Pulse sizing QC
- Qubit DNA concentration measurement QC
- Shearing with Megaruptor or g-Tubes
2. SMRTbell Express TPK 2.0 Library Construction
- Perform single-tube, addition-only reactions
- ~4.5 h library construction workflow followed by size-
selection
- Typical library yield ~50%
3. Size-select & Purify SMRTbell Library
- Perform size selection using SageELF for HiFi Variant
Detection or Blue Pippin for HiFi de novo Assembly
applications
- (Optional) alternative AMPure PB Size Selection method
is available for de novo Assembly applications (Note:
HiFi read lengths will be shorter)
4. Sequencing Preparation (CCS Sequencing Mode)
- Anneal Sequencing Primer, bind Polymerase, perform
AMPure PB complex cleanup
- Follow QRC for diffusion loading recommendations
- 2 hrs (≤20 kb) / 4 hrs (>20 kb) Pre-Extension Time; 30-
hour movie collection time (Sequel II System)
5. Analyze
- For variant detection analysis, can use GATK or Google
Deep Variant
- For large genome de novo assembly, can use Falcon-
Unzip or other third-party software
1
2
3
5
4
Day 1
Day 2
PROCEDURE & CHECKLIST – PREPARING HIFI SMRTBELL
LIBRARIES USING SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0
List of Required Materials and Equipment for Library Construction and Size Selection
LIBRARY CONSTRUCTION WORKFLOW RECOMMENDATIONS FOR
SPECIFIC HIFI SEQUENCING APPLICATIONS
- High-quality genomic DNA (gDNA) can be sheared using a Diagenode Megaruptor tool (up to ~100 kb
fragment size) or Covaris g-TUBEs (up to ~20 kb fragment size)
- Depending on project requirements, SMRTbell libraries are size-selected using a Sage Science
SageELF system (recommended for variant detection) or BluePippin system (recommended for de novo
assembly). Note: AMPure PB Bead size selection may optionally be used for de novo assembly
applications but may result in shorter mean HiFi read lengths (compared to using BP size selection)
LIBRARY CONSTRUCTION RECOMMENDATIONS FOR APPLICATIONS REQUIRING HIFI LONG READS
HIFI
APPLICATION
FOCUS
SIZE-SELECTION
METHOD
NUMBER OF COLLECTED
FRACTIONSNOTES
HiFi Variant
Detection
SageELF
(Recommended)
5 Fractions
(~11 kb, ~13 kb, ~15 kb, ~17 kb, >19 kb)
Resulting HiFi reads may also be used for de
novo assembly.
BluePippin2 Fractions
(~11-13 kb, ~13-20 kb)
Resulting HiFi reads may also be used for de
novo assembly.
HiFi de novo
Assembly
BluePippin (High-
Pass)
(Recommended)
1 Fraction
(>15 kb)
Removes <15 kb SMRTbells from final library
(with no upper-end MW cutoff).
Resulting HiFi reads are not recommended for
variant detection.
AMPure PB Beads1 Fraction
(>~5-20 kb, depending on shear distribution)
Removes <5 kb and reduces <10 kb SMRTbells
from final library
Resulting HiFi reads may show shorter HiFi
read lengths (compared to BP size selection)
and are not recommended for variant detection.
GENOMIC DNA SAMPLE INPUT & QUALITY REQUIREMENTS FOR
HIFI SEQUENCING
- This procedure describes construction of HiFi libraries from gDNA with a sheared mode size of >15 kb
- Table below summarizes DNA input, quality and DNA shear mode requirements for specific size-selection
options.
- The final SMRTbell library synthesis yield (%) of the collected and purified HiFi fractions depends on the
quality of the starting genomic DNA and distribution of the DNA shear.
- To increase the recovery yield of larger fraction sizes (>20 kb), the target shear size distribution
must be adjusted so that the mode is 20 kb or larger.
- Always perform test shears prior to starting SMRTbell library construction.
DNA INPUT REQUIREMENTS AND RECOMMENDED SHEARING METHODS FOR CONSTRUCTING HIFI SMRTBELL
LIBRARIES
Size-Selection
Method
Required Input gDNA
Amount
Required Input gDNA
Quality (Mode Size)
Target Sheared
Fragment Size
Distribution Mode*
Shearing Method**
SageElf 15 µg >40 kb >15 – 20 kb (or larger) g-TUBE or Megaruptor
BluePippin 15 µg >40 kb >15 – 20 kb (or larger) g-TUBE or Megaruptor
AMPure PB 15 µg >40 kb >20 kb g-TUBE or Megaruptor
* To increase the recovery yield of larger fraction sizes (>20 kb), the target shear size distribution must be adjusted so that the
mode is 20 kb or larger.
** gDNA can be sheared up to ~20 kb fragment size using Covaris g-TUBEs
1. CHEF Mapper XA System (Bio-Rad)
2. Femto Pulse System (Agilent)
3. Pippin Pulse System (Sage Science)
http://www.sagescience.com/products/pippin-pulse/
http://www.bio-rad.com/en-us/category/pulsed-field-gel-electrophoresis-systems
https://www.agilent.com/en/product/automated-electrophoresis/femto-pulse-
systems
➢ Up to 10 Mb
➢ >16 Hour Run Time
➢ Up to 80 kb
➢ 16 Hour Run Time
Recommended methods for determining gDNA size distribution:
Lane 1: 8-48 kb Ladder (Bio-Rad)
Lane 2: 5 kb Ladder (Bio-Rad)
Lane 3: HMW gDNA
Lane 4: Degraded gDNA
Lane 1: High MW gDNA
Lane 2: Degraded gDNA
Lane 3: 165 kb Ladder
Evaluation of gDNA quality using A) Bio-Rad CHEF Mapper and
B) Advanced Analytical Femto Pulse. Lanes A3 and B1 show an
example of a high quality, high molecular weight gDNA sample,
where most of the DNA migrates as a prominent band at the top of
the gel image. Lanes A4 and B2 show an example of a partially
degraded gDNA sample where most of the DNA migrates below ~40
kb and is thus not suitable for constructing a >15 kb HiFi SMRTbell
library.
A B
RECOMMENDATIONS FOR EVALUATING GENOMIC DNA QUALITY
➢ Up to 165 kb
➢ 1.5 Hour Run Time
Femto Pulse System only requires
~0.5 ng of gDNA sample and
assay run time is <1.5 hours
EXAMPLE SIZING QC ANALYSIS OF STARTING INPUT GENOMIC DNA
ISOLATED FROM A TREE PLANT SAMPLE
- Use these example size distribution profiles as guidance for determining the appropriate starting amount of
gDNA to use for this procedure. E.g., if your starting gDNA fragment size distribution is significantly lower in
quality than shown here, consider increasing the amount of starting input gDNA amount to be higher than
15 µg to help achieve optimal library construction recovery yields.
Size distribution mode of starting gDNA should be >40 kb to help achieve optimal
library construction recovery yields
Femto Pulse sizing QC of replicate Redwood tree plant genomic DNA samples. gDNA was isolated using the Circulomics Nanobind
Plant Nuclei Big DNA Kit (PN NB-900-801-01).
Unsheared
gDNA (160 kb Mode)
>40 kb
PERFORMING ACCURATE SIZING QC THROUGHOUT HIFI SMRTBELL
LIBRARY CONSTRUCTION IS IMPORTANT
SMRTbell library (17.3 kb Mode)
Sheared gDNA (19.8 kb Mode)
- Use these example size distribution profiles as guidance for determining the appropriate starting amount of
gDNA to use for this procedure
- To increase the recovery yield of larger HiFi SMRTbell insert sizes (>20 kb) during the size selection
step, the target shear size distribution must be adjusted so that the mode is 20 kb or higher.
Size distribution mode of sheared gDNA should be >15 kb to help achieve optimal
library construction recovery yields
Femto Pulse sizing QC of a sheared human gDNA and SMRTbell library sample. gDNA was sheared with the Megaruptor 1 System.
>15 kb
GENOMIC DNA SHEARING RECOMMENDATIONS FOR HIFI
SMRTBELL LIBRARY CONSTRUCTION
This Procedure & Checklist describes the construction of HiFi libraries from
gDNA with a target sheared mode size of >15 – 20 kb or larger
- Genomic DNA can be sheared using a Megaruptor System (Diagenode) or g-TUBEs*
(Covaris)
- Note: gDNA can be sheared up to ~20 kb fragment size using Covaris g-TUBEs
- If the goal of your project is to sequence larger HiFi library fraction sizes (>20 kb), the
shear size distribution must have a mode of 20 kb or larger.
- Megaruptor System is recommended if shearing gDNA to a target sheared mode size >20 kb
- The response of individual gDNA samples to recommended shearing parameters may differ,
so small-scale test shears are highly recommended.
- Under- or over-shearing gDNA will result in low yields of the final, size-fractionated library.
- For high quality gDNA, typical yields of sheared and concentration DNA are approx. ≥70%.
- Because you will need 10 μg of sheared gDNA for the subsequent enzymatic steps, PacBio
recommends starting the shearing procedure with at least 15 μg of input gDNA.
* See Appendix I of this technical training presentation for an overview
of an optional alternative shearing method using Covaris g-TUBEs to
shear gDNA samples to a target mode size of ≥15-20 kb.
MEGARUPTOR TOOL RECOMMENDATIONS FOR SHEARING
DNA FOR HIFI SMRTBELL LIBRARY CONSTRUCTION
Megaruptor generates a tight DNA shearing distribution profile that results in good
recoveries during SMRTbell library size selection
- Dilute 15 µg of high molecular weight gDNA in 1x Elution Buffer to a
concentration of 50 ng/µL in a volume of 300 µL
- It is important not to exceed this DNA concentration during shearing
or you may clog the hydropores
- Before shearing, remove a 1 µL aliquot (un-sheared sample) for QC
- Shear gDNA to the desired target mode size with long hydropores
using the recommended settings in the Megaruptor software as
shown in the Table below
DESIRED TARGET SHEAR
SIZE MODEMEGARUPTOR SYSTEM
MEGARUPTOR SUGGESTED
SOFTWARE SETTING*
11 – 13 kb Megaruptor 1 or 2 15 kb
15 – 18 kb Megaruptor 1 or 2 20 kb
21 – 23 kb Megaruptor 3 Speed 30
25 – 27 kb Megaruptor 3 Speed 28
* Perform small-scale test shears to verify appropriate Megaruptor software settings to achieve the desired target shear size mode for a specific sample.
Megaruptor 2 Megaruptor 3
RECOMMENDED MEGARUPTOR SOFTWARE SETTINGS FOR SHEARING GENOMIC DNA FOR HIFI LIBRARY CONSTRUCTION.
MEGARUPTOR TOOL RECOMMENDATIONS FOR SHEARING
DNA FOR HIFI SMRTBELL LIBRARY CONSTRUCTION (CONT.)
- Run time is approximately 15-20 minutes per tube using a Megaruptor 1 System.
- Proceed to the next step to concentrate your sheared gDNA using AMPure PB beads
- Note: The Megaruptor 1 tool dilutes DNA during shearing, so empirically measure the
volume of your sheared gDNA sample before AMPure purification
- Because the response of individual gDNA samples to recommended shearing parameters
may differ, small-scale test shears are highly recommended (e.g., 50 μL at 50 ng/μL)
- Under- or over-shearing gDNA will result in low yields of final, size fractionated SMRTbell
library
PERFORMING SHEARING OPTIMIZATIONS IS IMPORTANT
.
Sample 1
12.3 kb Mode
Sample 2
10.3 kb Mode
Sample 3
13.5 kb Mode
- Above Figure shows examples of 3 different gDNA samples sheared using the same Megaruptor 1 System
shearing protocol.
- Under- or over-shearing impacts yield of fractions recovered from the library size selection step, hence
impacting the number of SMRT Cells that can be achieved per library prep
Performing test shears is recommended since different genomic DNA samples may
shear differently
MEGARUPTOR 1 SYSTEM HUMAN GENOMIC DNA SHEARING
EXAMPLE
- Femto Pulse analysis of a human gDNA sample sheared to a 18 kb mode size using the ‘20 kb’ shear size
setting in the Megaruptor 1 software.
Sheared gDNA
(18.2 kb Mode)
MEGARUPTOR 3 SYSTEM BACTERIAL GENOMIC DNA SHEARING
EXAMPLE
- Femto Pulse analysis of replicate E. coli gDNA samples sheared using the ‘Speed 30’ setting in the
Megaruptor 3 software. Sizing QC results indicate that the gDNA was sheared to a ~22 – 23 kb mode size.
Sheared gDNA
(Mode = 22 – 23 kb)
MEGARUPTOR 3 SYSTEM PLANT GENOMIC DNA SHEARING
EXAMPLE
- Femto Pulse analysis of replicate Redwood tree plant gDNA samples sheared using the ‘Speed 28’ setting
in the Megaruptor 3 software. Sizing QC results indicate that the gDNA was sheared to a ~25 – 27 kb
mode size
Sheared gDNA
(Mode = 25 – 27 kb)
SageELF SYSTEM IS RECOMMENDED FOR HIFI LIBRARY SIZE
SELECTION FOR VARIANT DETECTION APPLICATIONS
- 12 size fractions are collected per SageELF gel cassette
(Run Time is ~4.5 hours)
- SMRTbell libraries with insert sizes of ~15 – 25 kb are
suitable for HiFi sequencing
- SageELF Cassette Definition File and Run Protocol Setup
(Pages 13 – 14 in Procedure)
- “0.75% 1-18kb v2”; ‘Size-based’ separation mode; Target
value = 3400 (move the bar slider to select Well #12)
- Recommended SMRTbell library input amount per lane: 5 µg
- For >5 µg, use a second gel cassette
- Typical recovery yields for each collected fraction of interest is ~≥10% of the total amount of library
material loaded per lane – but will be highly dependent on the size distribution of the starting sheared
gDNA
- Recommend performing DNA quantitation and sizing QC analyses for samples collected in
the following wells:
- 15 – 20 kb HiFi Libraries: Wells 3, 4, 5, 6 and 7
- >20 kb HiFi Libraries: Wells 1, 2, 3, 4, and 5
- Purify each size-selected fraction of interest with 0.5X AMPure PB Beads
- Final elution volume is 11 µL
- Perform final sizing QC for each purified fraction (e.g., by Femto Pulse, Fragment Analyzer,
CHEF Mapper or Pippin Pulse)
- Save desired fractions for sequencing (or store fractions at -20°C for future use)
* See Appendix II of this presentation for an overview of an optional alternative
size selection method using BluePippin (Sage Science) for variant detection
applications.
SageELF SYSTEM IS RECOMMENDED FOR HIFI LIBRARY SIZE
SELECTION FOR VARIANT DETECTION APPLICATIONS (CONT.)
SageELF SYSTEM IS RECOMMENDED FOR HIFI LIBRARY SIZE
SELECTION FOR VARIANT DETECTION APPLICATIONS (CONT.)
Left figure shows an overlay of Femto Pulse electropherograms for SageELF Fractions 2, 3, 4 and 5 collected for a human library
sample. Fraction 3 contains size-selected SMRTbell templates with a mean insert size of ~15 kb.
F2
F3
F4F5
F3
Fraction 3(15.2 kb Mode)
F4 F2F5
15.2 kb10.7 kb 17.9 kb9.4 kb
Always perform sizing QC on recovered SMRTbell library fractions after size-
selection
- To determine which fractions are suitable for generating HiFi reads, perform a sizing QC on
all recovered SMRTbell library fractions of interest after size selection using Femto Pulse,
Fragment Analyzer, CHEF Mapper or Pippin Pulse
- Femto Pulse sizing QC method is highly recommended since it requires very small amounts of DNA
sample (≤500 pg)
- SMRTbell libraries with insert sizes of ~15 – 25 kb are suitable for HiFi (CCS) sequencing
SIZE SELECTION WITH THE BLUEPIPPIN SYSTEM
- BluePippin size selection protocol options for HiFi library construction:
- Option 1: Size-select a single fraction (>15 kb) – Recommended size-selection method for de
novo assembly applications [see Slides 41 – 44 in this presentation for description of procedure]
- Option 2: Size-select two fractions (9 – 13 kb and >15 kb) – Can be used to size-select HiFi
libraries for variant detection applications if a Sage Science SageELF system is unavailable [see
following Slides 67 – 71 in Appendix II of this presentation for description of procedure]
- For the latest BluePippin User Manual and guidance on size-selection protocols, please
contact Sage Science (www.sagescience.com)
- Note: Visit Sage's website (http://www.sagescience.com) to verify that your BluePippin
software is up-to-date.
As an alternative to the SageELF system, Sage Science’s BluePippin tool may also
be used to size-select libraries for HiFi sequencing
BLUEPIPPIN SYSTEM IS RECOMMENDED FOR HIFI LIBRARY SIZE
SELECTION FOR DE NOVO ASSEMBLY APPLICATIONS
1. Prepare up to 5 µg of SMRTbell templates in a final volume of 30 μL Elution Buffer for
each Blue Pippin lane.
2. Bring the Loading Solution to room temperature, and then add 10 μL of the Loading
Solution to the 30 μL DNA sample. For loading multiple lanes with the same sample,
scale the volumes proportionally. The Loading Solution is viscous, so pipet slowly to
ensure complete transfer into the DNA sample.
a) Pipette mix using wide-bore pipette tips to mix.
b) Spin briefly to collect the contents at the bottom of the tube.
3. Follow the manufacturer’s recommendations to set up a run protocol.
a) Select the “0.75% DF Marker S1 high-pass 15 kb -20kb” Cassette Definition File for your
sample.
The following BluePippin High-Pass procedure may be used to size-select a single
fraction (>15 kb) for de novo assembly applications using HiFi sequencing
BLUEPIPPIN SYSTEM IS RECOMMENDED FOR HIFI LIBRARY SIZE
SELECTION FOR DE NOVO ASSEMBLY APPLICATIONS (CONT.)
3. (Cont.)
b) Using the “Range” selection mode, enter a desired “BPstart” value (e.g., between 15 kb and 20 kb).
A “BP End” value should automatically appear.
Note: Sample lanes containing <3 µg of SMRTbell library material will run faster during
electrophoresis. In such cases, PacBio recommends adjusting the BP Start values as follows:
* PacBio does not recommend running BluePippin size selection with <2 µg of SMRTbell library material per lane.
c) Be sure to assign a marker lane. We recommend using Lane 4 for the marker.
4. Load the S1 marker and samples into the BluePippin gel cassette and start the run. Run
time is approximately 4.5 hours.
5. Collect the size-selected library: To maximize recovery of eluted DNA, wait at least 20
minutes after the run terminates before removing the sample from the elution chamber.
a. Collect the eluate containing the size-selected library into a 2.0 mL DNA LoBind tube.
b. Wash the elution well with 40 μL of Sage Science’s 0.1% Tween-20 Wash Solution and then add the
recovered wash liquid to the eluted sample. Washing the elution well may further increase recovery
yields by approximately 10-20%.
CASSETTE DEFINITION FILE IF < 3 µg INPUT PER LANE*, USE BP START
0.75% DF Marker S1 high-pass 15 kb -20kb
12500 for 15 kb cutoff
13500 for 17 kb cutoff
15000 for 20 kb cutoff
BLUEPIPPIN SYSTEM IS RECOMMENDED FOR HIFI LIBRARY SIZE
SELECTION FOR DE NOVO ASSEMBLY APPLICATIONS (CONT.)
Sheared gDNAMode = 27 kb
SMRTbell Library
(Pre-Size Selection)Mode = 24 – 25 kb
Example Femto Pulse Sizing QC for >17 kb BluePippin Size-Selected HiFi Libraries
SMRTbell Library
(Post-BP Size Selection)Mode = 23 – 25 kb
Femto Pulse sizing QC analysis for replicate Redwood tree plant library samples size-selected on the BluePippin system using
the “0.75% DF Marker S1 high-pass 15 kb -20kb” cassette definition file with a >17 kb lower molecular weight cutoff. Most
SMRTbell templates <17 kb are removed after BluePippin size-selection while templates >50 kb still remain.
ESTIMATED RECOVERY YIELDS FOR HIFI LIBRARY CONSTRUCTION
WORKFLOW STEPS
WORKFLOW STEPESTIMATED
YIELD
Average Post Shearing Yield (from starting gDNA) ~70 %
Average SMRTbell Yield (from sheared gDNA input into Remove ssDNA Overhangs step)
~50 %
Average Yield of ~15 kb SageELF Size-Selected Fraction (from input DNA into SageELF System)
~≥10 %*
Average yield of >15 kb BluePippin Size-Selected Fraction (from input DNA into BluePippin System)
~10 %*
* HiFi library yield after size selection is highly dependent on the final SMRTbell library size distribution
HiFi Library Sequencing Workflow Details
SAMPLE SETUP RECOMMENDATIONS FOR HIFI SMRTBELL
EXPRESS TPK 2.0 LIBRARIES – SEQUEL II SYSTEM (CHEMISTRY 2.0)
- Follow SMRT Link Sample Setup instructions using the
recommendations provided in the Quick Reference Card –
Loading and Pre-Extension Time Recommendations for
the Sequel II System*
* Sequence each size-selected fraction of interest individually
HiFi Sequencing Performance Example
Data
HIFI SMRTBELL EXPRESS TPK 2.0 LIBRARY EXAMPLE 1:SAGEELF SIZE-SELECTION LIBRARY RECOVERY YIELDS FOR HUMAN
GENOMIC DNA SAMPLE SHEARED TO 15 KB MODE SIZE
Pre-Size Selection
DNA Input Into
Shearing
DNA Input Into
Library Construction
DNA Input
Into SageELF
SageELF Fraction
Average Size
Size-Selected
Library Recovery
Amount (%)
9600 ng 9153 ng 3720 ng Frac 3 – 22.4 kb 568 ng (~15%)
Frac 4 – 16.6 kb 696 ng (~19%)
Frac 5 – 16.0 kb 586 ng (~16%)
Frac 6 – 12.1 kb 378 ng (~10%)
Frac 7 – 11.3 kb 240 ng (~6%)
15 kb ModeFraction of interest
(Frac 4 = 16.8 kb Mode)
Post-Size Selection
HIFI SMRTBELL EXPRESS TPK 2.0 LIBRARY EXAMPLE 1 (CONT.):PRIMARY SEQUENCING METRICS PERFORMANCE
Sample Name StatusMovie Time
(hours)
Pre-
extension
Time
(hours)
Total Bases
(Gb)
Unique
Molecular
Yield (Gb)
Read Length Productivity
Polymerase Longest SubreadP0 P1 P2
Mean N50 Mean N50
1Frac_4 15 kb HiFi Library
Complete 30 2 392.68 56.88 91960 181775 14514 15649 44.6% 53.3% 2.1%
HiFi data shown were generated using Sequel II Chemistry 2.0EA
15 kb Human HiFi Express 2.0 Library Achieved ~400 Gb Total Bases and >90 kb
Polymerase Read Length
HIFI SMRTBELL EXPRESS TPK 2.0 LIBRARY EXAMPLE 1 (CONT.):HIFI (CCS) BASE YIELD PERFORMANCE
HiFi data shown were generated using Sequel II Chemistry 2.0EA
15 kb Human HiFi Express 2.0 Library Generated 1.8 Million ≥Q20 CCS Reads and
27 Gb of ≥Q20 CCS Bases
CCS ANALYSIS METRIC 15 KB HUMAN HIFI LIBRARY
HiFi (≥Q20) Reads 1,846,154
HiFi (≥Q20) Base Yield 27.3 Gb
HiFi (≥Q20) Read Length (Mean) 14,773 bp
HiFi (≥Q20) Read Quality (Median) Q32
Read Quality DistributionRead Length Distribution
HIFI SMRTBELL EXPRESS TPK 2.0 LIBRARY EXAMPLE 2:SAGEELF SIZE SELECTION LIBRARY RECOVERY YIELDS FOR E. COLI
GENOMIC DNA SAMPLE SHEARED TO 21 KB MODE SIZE
Pre-Size Selection
DNA Input Into
Shearing
DNA Input Into
Library Construction
DNA Input
Into SageELF
SageELF Fraction
Mode Size
Size-Selected
Library Recovery
Amount (%)
12,000 ng 10,000 ng 2900 ng Frac 1 – 28.8 kb 27.8 ng (~1%)
Frac 2 – 25.8 kb 904 ng (~31%)
Frac 3 – 19.4 kb 426 ng (~15%)
Frac 4 – 15.3 kb 115 ng (~4%)
Frac 5 – 13.2 kb 72.4 ng (~3%)
Mode = 21 kb Fraction of interest
(Frac 2 Mode = 25.8 kb)
Post-Size Selection
HIFI SMRTBELL EXPRESS TPK 2.0 LIBRARY EXAMPLE 2 (CONT.):PRIMARY SEQUENCING METRICS PERFORMANCE
Sample Name StatusMovie Time
(hours)
Pre-
extension
Time
(hours)
Total Bases
(Gb)
Unique
Molecular
Yield (Gb)
Read Length Productivity
Polymerase Longest SubreadP0 P1 P2
Mean N50 Mean N50
1Frac_2 25 kb HiFi Library
Complete 30 4 415.3 114.4 87797 169050 24791 29168 39.2 59.1 1.8
2Frac_3 20 kb HiFi Library
Complete 30 4 447.8 87.3 98645 180352 19929 21108 41.9 56.7 1.5
HiFi data shown were generated using Sequel II Chemistry 2.0
≥20 kb E. coli HiFi Express 2.0 Libraries Achieved >400 Gb Total Bases and >85 kb
Polymerase Read Length
50 kb
20 kb
25 kb HiFi Library20 kb HiFi Library
50 kb
20 kb
HIFI SMRTBELL EXPRESS TPK 2.0 LIBRARY EXAMPLE 2 (CONT.):HIFI (CCS) BASE YIELD PERFORMANCE
HiFi data shown were generated using Sequel II Chemistry 2.0
>20 kb Human HiFi Express 2.0 Libraries Generated 1.1 – 1.8 Million ≥Q20 CCS
Reads and 30 – 36 Gb of ≥Q20 CCS Bases
CCS ANALYSIS METRIC 20 KB HUMAN HIFI LIBRARY 25 KB HUMAN HIFI LIBRARY
HiFi (≥Q20) Reads 1,836,137 1,129,580
HiFi (≥Q20) Base Yield 36 Gb 30 Gb
HiFi (≥Q20) Read Length (Mean) 19,683 bp 26,149 bp
HiFi (≥Q20) Read Quality (Median) Q29 Q25
Read Quality DistributionRead Length Distribution
20 kb HiFi Library
Read Quality DistributionRead Length Distribution
25 kb HiFi Library
HiFi Sequencing Data Analysis
Recommendations for Variant Detection
and de novo Assembly Applications
HIFI SEQUENCING DATA ANALYSIS RECOMMENDATIONS FOR
VARIANT DETECTION APPLICATIONS
- Utilize SMRT Link to generate highly accurate (≥Q20) single-molecule reads (HiFi reads)
using the Circular Consensus Sequencing (CCS) analysis application
- Output data in standard file formats, (BAM and FASTA/Q) for seamless integration with
downstream analysis tools
- Can use third-party software for variant detection analysis (SNVs, InDels and SVs) using HiFi
reads generated via CCS Sequencing Mode:
- GATK
- Google Deep Variant
- Note: For structural variation detection analysis (SVs ≥20 bp) using standard Long Reads
generated via CLR Sequencing Mode, can use Structural Variant Calling analysis application
in SMRT Link or pbsv command line tools.
- Contact PacBio Technical Support ([email protected]) or your local Bioinformatics Field
Applications Scientist for additional information about data analysis recommendations
HiFi reads are compatible with third-party variant calling tools
HIFI SEQUENCING DATA ANALYSIS RECOMMENDATIONS FOR DE
NOVO ASSEMBLY APPLICATIONS
- Utilize SMRT Link to generate highly accurate (≥Q20) single-molecule reads (HiFi reads)
using the Circular Consensus Sequencing (CCS) analysis application
- Output data in standard file formats, (BAM and FASTA/Q) for seamless integration with
downstream analysis tools
- Can use PacBio DevNet Tools or other third-party software for de novo assembly analysis
using HiFi reads generated via CCS Sequencing Mode:
- FALCON / FALCON-Unzip
- Hifiasm
- HiCanu
- Peregrine
- Contact PacBio Technical Support ([email protected]) or your local Bioinformatics Field
Applications Scientist for additional information about data analysis recommendations
HiFi reads are compatible with third-party de novo assembly tools
Technical Documentation & Applications
Support Resources
BEST PRACTICES: VARIANT DETECTION USING WHOLE GENOME
SEQUENCING WITH HIFI READS (SEQUEL II CHEMISTRY 2.0)
SMRTbell Template Preparation
- Start with unamplified genomic DNA input (≥15 µg)
- Prepare a HiFi library for using SMRTbell Express Template Prep Kit 2.0
- Enrich for 15 – 20 kb inserts with size selection
Sequence on the Sequel II System (CCS Sequencing Mode)
- Maximize output and turn-around-time with adjustable sequencing parameters
- Sequence to desired coverage based on study needs:*
- Recommend 2 SMRT Cells 8M to achieve 15-fold coverage of a human genome for comprehensive
variant detection for $2600†
Data Analysis Solutions with the PacBio Analytical Portfolio
- Detect all variant types – including SNVs, indels, SVs, and CNVs – with the highest precision
and recall using SMRT Analysis, and GATK or Google DeepVariant
- Google DeepVariant achieves higher precision and recall than GATK, particularly for indels
- Expand variant calling into previously inaccessible regions of the genome, including
repetitive regions and medically relevant genes that are difficult to map
- Output data in standard file formats – BAM and VCF – for seamless integration with
downstream analysis tools
- Phase small variants into phase blocks using WhatsHap
- Confirm variant calls visually with IGV10 and GenomeRibbon
* Read lengths, reads/data per SMRT Cell 8M and other sequencing performance results vary based on sample quality/type and insert size.† Prices, listed in USD, are approximate and may vary by region. Pricing includes library and sequencing reagents run on a Sequel II System and does not include instrument
amortization or other reagents.
BEST PRACTICES: WHOLE GENOME SEQUENCING (WGS) FOR
DE NOVO ASSEMBLY (SEQUEL II CHEMISTRY 2.0)
-HiFi reads provide a good balance of
read length (up to 25 kb) and accuracy
(≥99%)
- Long reads to span small repeats
- High accuracy to see minute differences
between long repeats
- Small file sizes and fast analysis time
- Lower coverage requirements for high-
quality results
- Contig N50
- Accuracy
- Genes in frame
- Phasing
- Assembly size
- Gene completeness
(BUSCO)
Contiguity
Completeness
Correctness
BEST PRACTICES: WHOLE GENOME SEQUENCING (WGS) FOR
DE NOVO ASSEMBLY (SEQUEL II CHEMISTRY 2.0) (CONT.)
- Start with >15 µg DNA
~40 kb
- Use HiFi Express
Template Prep Kit 2.0
- Size select at either >15
kb or >20 kb
- Sequence in Circular
Consensus Sequencing
(CCS) mode
- Sequence to 10-fold
coverage per haplotype
for phased assembly
- Generate HiFi reads
with CCS analysis
(command line or SMRT
Link)
- Assemble and phase
HiFi reads with
FALCON & FALCON-
Unzip or wide array of
community tools
1 2 3
TECHNICAL DOCUMENTATION & APPLICATIONS SUPPORT
RESOURCES FOR HIFI SEQUENCING
- Sequel II System Operations Guide (PN 101-774-700)
- Quick Reference Card – SMRT Link v8.0 Migration (PN 101-850-100)
- Sequel II System 8.0 Release Notes (PN 101-774-400)
- SMRT Link v8.0 Software Download Site: https://www.pacb.com/support/software-downloads/
- SMRT Link v8.0 Software Installation Instructions (PN 100-749-900)
- SMRT Link v8.0 Release Notes (PN 100-749-600)
- SMRT Link v8.0 User Guide (PN 101-039-100)
- SMRT Tools Reference Guide (v8.0) (PN 100-939-900)
- Sequel SMRT Link Web Services API Use Cases (v8.0) (PN 101-430-800)
- Quick Reference Card – Loading and Pre-Extension Recommendations for the Sequel II System (PN 101-
769-100)
- Pacific Biosciences Glossary of Terms (PN 000-710-267)
Sequel II System and SMRT Link Documentation
https://www.pacb.com/support/documentation/
TECHNICAL DOCUMENTATION & APPLICATIONS SUPPORT
RESOURCES FOR HIFI SEQUENCING (CONT.)
- Procedure & Checklist – Preparing HiFi SMRTbell Libraries using SMRTbell Express Template Prep Kit
2.0 (PN 101-853-100)
- Procedure & Checklist - Preparing SMRTbell Libraries Using Express Template Prep Kit 2.0 With Low
DNA Input (PN 101-730-400)
- Procedure & Checklist – Using AMPure PB Beads for Size-Selection (PN 101-854-900)
- Application Brief: Variant Detection using Whole Genome Sequencing With HiFi Reads – Best Practices
(PN BP106-092419)
- Application Brief: Whole Genome Sequencing (WGS) for De novo Assembly – Best Practices (PN
BP102-121219)
- Overview – Sequel Systems Application Options and Sequencing Recommendations (PN 101-851-300)
https://www.pacb.com/support/documentation/
Sample Library Preparation Protocol Documentation & Application Briefs
Appendix I: Optional Alternative Shearing
Method Using Covaris g-TUBEs
DNA SHEARING USING COVARIS G-TUBES
Specific guidance for shearing gDNA to ≥15 – 20 kb using the Covaris g-TUBE
(modified from the Covaris User Manual) is provided below and should be strictly
followed to achieve the desired target fragment size.
1. Dilute 15 µg high molecular weight gDNA in 1X Elution Buffer to a concentration of 83.3
ng/µL in a volume of 180 µL.
2. Transfer gDNA to g-TUBE and shear the gDNA at 2029 x g (5500 rpm in the Eppendorf
MiniSpin Plus) for 2 minutes.
3. Check for any residual sample remaining in the upper chamber. If present, re-spin for
another 2 minutes. Repeat spin until entire gDNA sample has passed through the orifice.
4. Invert and spin 2029 x g (5500 rpm in the Eppendorf MiniSpin Plus) until entire gDNA
sample has passed through the orifice.
5. Repeat step 4, FOUR times for a total of SIX passes through the orifice.
6. Transfer the sheared gDNA to a new 2.0 mL DNA Lo-bind microfuge tube.
7. Proceed to the “Concentrate DNA using AMPure PB Beads” section in the Procedure &
Checklist to concentrate your sheared gDNA using AMPure beads.
SIZING QC OF GENOMIC DNA SHEARED USING g-TUBES
- Femto Pulse sizing QC of sheared gDNA shows a mode of ~20 kb
Appendix II: Optional Alternative Size-
selection Method Using the Sage Science
BluePippin System for Variant Detection
Applications
SIZE SELECTION WITH THE BLUEPIPPIN SYSTEM
- BluePippin size selection protocol options for HiFi library construction:
- Option 1: Size-select a single fraction (>15 kb) – Recommended size-selection method for de
novo assembly applications [see Slides 41 – 44 in this presentation for description of procedure]
- Option 2: Size-select two fractions (9 – 13 kb and >15 kb) – Can be used to size-select HiFi
libraries for variant detection applications if a Sage Science SageELF system is unavailable [see
Slides 67 – 71 in Appendix II of this presentation for description of procedure]
- For the latest BluePippin User Manual and guidance on size-selection protocols, please
contact Sage Science (www.sagescience.com)
- Note: Visit Sage's website (http://www.sagescience.com) to verify that your BluePippin
software is up-to-date.
As an alternative to the SageELF system, Sage Science’s BluePippin tool may also
be used to size-select libraries for HiFi sequencing
OPTIONAL ALTERNATIVE BLUEPIPPIN SIZE SELECTION PROTOCOL: SIZE-SELECT TWO FRACTIONS (9 – 13 KB AND >15 KB) FOR VARIANT
DETECTION APPLICATIONS
1. Prepare up to 5 µg of SMRTbell templates in a final volume of 30 μL Elution Buffer for
each Blue Pippin lane.
2. Bring the Loading Solution to room temperature, and then add 10 μL of the Loading
Solution to the 30 μL DNA sample. For loading multiple lanes with the same sample, scale
the volumes proportionally. The Loading Solution is viscous, so pipet slowly to ensure
complete transfer into the DNA sample.
a) Pipette mix using wide-bore pipette tips to mix.
b) Spin briefly to collect the contents at the bottom of the tube.
3. Follow the manufacturer’s recommendations to set up a run protocol.
a) Select the “0.75% DF Marker S1 3-10 kb Improved Recovery” Cassette Definition File for your
sample.
b) Using the “Range” selection mode, enter a desired “BPstart” value of 9000 and a BP End value of
13000.
Note: Sample lanes containing <3 µg of SMRTbell library material will run faster during
electrophoresis. In such cases, PacBio recommends adjusting the BP Start value to 8000 bp and
BP End value to 12000 bp. We do not recommend running lanes with <2 µg of SMRTbell library
material.
c. Be sure to assign a marker lane. We recommend using Lane 4 for the marker.
OPTIONAL ALTERNATIVE BLUEPIPPIN SIZE SELECTION PROTOCOL: SIZE-SELECT TWO FRACTIONS (9 – 13 KB AND >15 KB) FOR VARIANT
DETECTION APPLICATIONS (CONT.)
4. Load the S1 marker and samples into the BluePippin gel cassette and start the run. Run
time is approximately 4.5 hours.
5. Collect the 9-13 kb fraction: To maximize recovery of eluted DNA, wait at least 20
minutes after the run terminates before removing the sample from the elution chamber.
a. Collect the eluate containing the 9-13 kb fraction into a 2.0 mL DNA LoBind tube.
b. Wash the elution well with 40 µL of Sage Science’s Electrophoresis Buffer and add the recovered
wash liquid to the eluted sample. A second wash may further increase recovery yields.
c. Refill the elution chamber with 40 µL Electrophoresis Buffer and re-seal the chamber. Close the lid
of the BluePippin.
6. Collect the ≥15 kb fraction: Use Manual Mode to collect a second fraction.
a) From the Main Tab, select “Manual Mode” from the top of the “Protocol Name” drop down menu.
b. Click “Start” on the controller.
c. Click “Elute” box for each sample lane. Box will turn orange and current will be
detected.
d. When elution timer reaches 30 minutes, click “Idle” box for each sample lane. Idle box will become
white and current will drop.
e. Collect second eluate as described in Step 5 above, including washes. Typical insert size for this
manually eluted fraction is ~15kb.
7. Proceed to the final 0.5X AMPure PB Bead purification step in the Procedure & Checklist,
or store the collected fractions at -20°C for future use.
OPTIONAL ALTERNATIVE BLUEPIPPIN SIZE SELECTION PROTOCOL: SIZE-SELECT TWO FRACTIONS (9 – 13 KB AND >15 KB) FOR VARIANT
DETECTION APPLICATIONS (CONT.)
OPTIONAL ALTERNATIVE BLUEPIPPIN SIZE SELECTION PROTOCOL: SIZE-SELECT TWO FRACTIONS (9 – 13 KB AND >15 KB) FOR VARIANT
DETECTION APPLICATIONS (CONT.)
COLLECTED
FRACTION
MEAN SIZE
(KB)
1 12.2
2 19.2
BP Fraction 1(Mode = 12.6 kb)
BP Fraction 2(Mode = 17.7 kb)
Example Femto Pulse Sizing QC for 12 kb & 19 kb BluePippin Size-Selected HiFi
Library Fractions
Femto Pulse sizing QC analysis for a human library sample size-selected on the BluePippin system using the “0.75% DF Marker
S1 3-10 kb Improved Recovery” cassette definition file to collect two size fractions (9 – 13 kb and >15 kb). Femto Pulse smear
analyses show that that the mean SMRTbell library size for Fraction 1 is 12.2 kb and the mean size for Fraction 2 is 19.2 kb.
Appendix III: Optional Alternative Size-
selection Method Using AMPure PB Size-
Selection for De Novo Assembly
Applications
- Procedure & Checklist (PN 101-854-900) provides instructions
for using AMPure PB beads for size-selection of SMRTbell
libraries
- Note: AMPure PB Bead size-selection is not the primary
recommendation for HiFi de novo assembly applications since
it will result in shorter HiFi read lengths compared to using a
BluePippin system. AMPure PB Bead size-selection may,
however, be a useful option when sample quantity is limited.
- AMPure PB beads are diluted with PacBio Elution Buffer to
achieve a specified volumetric ratio and then used for size-
selection
- Important: To efficiently remove SMRTbell templates <3 kb or
<5 kb, be sure to use this procedure after the first AMPure PB
bead purification step (post-adapter ligation) in the HiFi
SMRTbell library construction workflow.
- Protocol document contains:
1. List of required materials
2. Procedure for diluting AMPure PB Beads in Elution Buffer
to achieve a specified volumetric concentration
3. Procedure for performing size selection to remove <3 kb
SMRTbell templates or <5 kb SMRTbell templates using
a specified volumetric ratio of diluted AMPure PB
Beads:Sample
PROCEDURE & CHECKLIST – USING AMPURE PB BEADS FOR SIZE-
SELECTION
Optional alternative AMPure PB size-selection protocol for use with HiFi de novo
assembly applications
https://www.pacb.com/support/documentation/
LIST OF REQUIRED MATERIALS AND EQUIPMENT FOR AMPURE PB
SIZE SELECTION PROCEDURE
To help minimize shearing-induced damage to SMRTbell templates, use wide-bore
pipette tips for pipette mixing AMPure PB Bead/DNA solutions
DILUTION OF AMPURE PB BEADS WITH ELUTION BUFFER
The final AMPure PB bead concentration is critical to the success of this procedure.
Therefore, accurate pipetting is of utmost importance to achieve a final 35% (v/v)
AMPure PB Bead working solution in Elution Buffer
SIZE-SELECTION PROCEDURE USING DILUTED AMPURE PB BEADS
Key Considerations
- Important: To efficiently remove SMRTbell templates <3 kb or <5 kb from your HiFi library
sample, be sure to use this procedure after the first AMPure PB bead purification step
(post-adapter ligation) in the SMRTbell library construction workflow.
- For effective size-selection using AMPure PB beads, accurate pipetting is necessary.
- Additionally, the DNA concentration of the SMRTbell library to be size-selected must be
0.5–10 ng/μL.
- Use of higher concentrations (>15-100 ng/μL) decreases the efficiency of reduction of short insert
SMRTbell templates. Adjust the sample concentration so that the DNA concentration is within this
range.
- Note: that presence of ethanol in the sample impacts yield - therefore it is important to
remove residual ethanol in the library prior to performing the AMPure PB size selection
procedure
- The volume of diluted AMPure PB beads to use for size-selection depends on the desired
molecular weight cutoff (see next slide).
- To remove <3 kb SMRTbell templates, use 3.7X ratio of diluted AMPure PB beads:sample.
- To remove <5 kb SMRTbell templates, use 3.1X ratio of diluted AMPure PB beads:sample.
- Note: It is critical to mix precise volumes of the sample and diluted AMPure PB beads to achieve
successful size-selection.
- SMRTbell library recovery yields with AMPure PB size-selection are typically >40%
SIZE-SELECTION PROCEDURE USING DILUTED AMPURE PB BEADS
(CONT.)
AMPure PB Size Selection Performance Is Dependent on the Volumetric Ratio of
Diluted AMPure PB Beads:Sample Used
- PacBio recommends using a 3.7X ratio of diluted AMPure PB Beads:Sample to remove
<3 kb SMRTbell templates and a 3.1X ratio to remove <5 kb SMRTbell templates.
Diluted AMPure PB Bead:Sample Volumetric Ratio
Siz
e (
bp
)
Figure shows the performance of diluted (35% v/v) AMPure PB beads for size-selection of a DNA sample
with a concentration of 8 ng/μL at varying ratios of diluted AMPure PB bead Volume:Sample Volume.
HIFI DE NOVO ASSEMBLY EXAMPLE USING AMPURE PB SIZE
SELECTION: DROSOPHILA GENOME ASSEMBLY
SampleSageELF
19 kb Fraction
SageELF
24 kb FractionAMPure PB SS
AMPure PB SS+15 kb In silico Cut
HiFi Base Yield
(Coverage)
31 Gb
(221-fold)
26 Gb
(186-fold)
33 Gb
(236-fold)
28 Gb
(200-fold)
Assembly Coverage
(Down-sampled)20-fold 20-fold 20-fold 20-fold
Assembly Length 156 Mb 160 Mb 165 Mb 163 Mb
Contig N50 6.46 Mb 9.30 Mb 4.27 Mb 5.71 Mb
N Contigs 332 278 349 297
AMPure PB size-selected HiFi library generated Drosophila genome assemblies
comparable in quality to SageELF size-selected HiFi libraries
General Recommendations for High-
Molecular Weight gDNA Isolation, QC &
Handling for SMRTbell Library Construction
Human
Biomedical
Research
Plant & Animal
Sciences
Microbiology
& Infectious
Disease
- If gel purification is required, avoid using ethidium/UV based visualization methods. One
alternative is to use SYBR® Safe (Invitrogen) and visualize with blue light
- To help resuspend the DNA, carefully invert the tube several times after adding buffer
and/or tap the tube gently. Avoid vortexing genomic DNA when possible as vortexing can
cause shearing of the DNA. It is also recommended to use wide bore tips in sample
handling
- Alternatively, allow the DNA to stand in buffer overnight at 25°C to resuspend
- Overheating can introduce DNA damage. Inactivate DNAase as recommended by the
vendor kit. It is best to avoid heat inactivation when possible
- DNA storage conditions: 4 °C (short-term); -20°C / -80°C (long-term)
GENERAL RECOMMENDATIONS FOR ISOLATING HIGH-MOLECULAR
WEIGHT GENOMIC DNA
EXAMPLE THIRD-PARTY HIGH-MOLECULAR WEIGHT GENOMIC
DNA ISOLATION PROTOCOL AND KIT SOLUTIONS
Note: The products below have not been extensively tested or validated by PacBio
R&D but are listed here as examples of third-party kits or methods used by other
PacBio customers for isolating genomic DNA for SMRTbell library preparation
Plant Tissue
QIAGEN Genomic-tip 20/100/500/G Kit
Unsupported Protocol – Switchgrass (Panicum virgatum) DNA isolation [USDA]
- http://www.pacb.com/wp-content/uploads/2015/09/Switchgrass-DNA-isolation.pdf
Unsupported Protocol – DNA extraction of Chlamydomonas using CTAB [JGI]
- http://www.pacb.com/wp-content/uploads/2015/09/DNA-extraction-chlamy-CTAB-JGI.pdf
QIAGEN User-Developed Prototocol: Isolation of genomic DNA from plants and filamentous fungi
using the QIAGEN Genomic-tip Kit
- https://www.qiagen.com/de/resources/resourcedetail?id=cb2ac658-8d66-43f0-968e-
7bb0ea2c402a&lang=en
Circulomics Nanobind Plant Nuclei Big DNA Kit
- https://www.circulomics.com/
Insect Tissue
QIAGEN User-Developed Prototocol: Isolation of genomic DNA from mosquitoes or other insects using
the QIAGEN Genomic-tip Kit
- https://www.qiagen.com/ca/resources/resourcedetail?id=b45c3cc3-7f2b-4f4a-aa37-
21d814ed3730&lang=en
Circulomics Nanobind Tissue Big DNA Kit
- https://www.circulomics.com/
Fish Tissue
QIAGEN Genomic-tip 20/100/500/G Kit
- Reference: Chromosomal-Level Assembly of the Asian Seabass Genome Using Long Sequence Reads and Multi-layered
Scaffolding. PLOS Genetics 12(4): e1005954. (2016) doi: 10.1371/journal.pgen.1005954
http://journals.plos.org/plosgenetics/article?id=10.1371%2Fjournal.pgen.1005954
QIAGEN User-Developed Prototocol: Purification of archive-quality DNA from 10–20 mg fish tissue
using the Gentra® Puregene® Tissue Kit or Gentra Puregene Mouse Tail Kit
- https://www.qiagen.com/ca/resources/resourcedetail?id=948e5a5e-57c4-482f-a852-
43ec3da97fce&lang=en
Circulomics Nanobind Tissue Big DNA Kit
- https://www.circulomics.com/
Human Tissue
QIAGEN Genomic-tip 20/100/500/G Kit / QIAGEN Gentra Puregene Cell Kit
Unsupported Protocol – Gentra Puregene Cell Kit (Qiagen) DNA Isolation [Univ. Washington]
- http://www.pacb.com/wp-content/uploads/2015/09/Gentra-Puregene-Qiagen-DNA-Isolation.pdf
Reference: Resolving the complexity of the human genome using single-molecule sequencing. Nature 517, 608-611 (29
January 2015) doi:10.1038/nature13907 http://www.nature.com/nature/journal/v517/n7536/full/nature13907.html
Circulomics Nanobind CBB Big DNA Kit or Nanobind Tissue Big DNA Kit
- https://www.circulomics.com/
Macherey-Nagel™ NucleoBond™ AXG 20/100/500 Gravity-flow Columns
- https://www.mn-net.com/
NEW SAMPLE PREPARATION ONLINE RESOURCE
Literature resource for sample collection and DNA extraction protocol references
- PacBio does not assume responsibilities/guarantees for these external publications/protocols, but we are
happy to help as best as we can to guide/connect. Please contact [email protected] for more
discussions around your particular species & sequencing project!
www.ExtractDNAforPacBio.com
High Molecular Weight gDNA and SMRTbell Library Cleanup
AMPure PB Bead Wash
- Refer to the appropriate Procedure & Checklist protocol document for specific recommendations for AMPure PB bead
purification of different SMRTbell library insert sizes
GENERAL RECOMMENDATIONS TO HELP CLEAN UP HIGH
MOLECULAR WEIGHT GENOMIC DNA AND SMRTBELL LIBRARIES
Starting with high-quality, high molecular weight (HMW) genomic DNA will result
in longer libraries and better de novo assembly performance
METHODS FOR EVALUATION OF GENOMIC DNA QUALITY
- Input genomic DNA must be carefully QC’d to assess integrity
- PFGE/FIGE or Femto Pulse sizing tool is highly recommended
- High molecular weight DNA → long read lengths
- Degraded DNA → short read lengths, low library recovery yields (dependent on size selection
parameters)
- DNA purity can be determined by using a NanoDrop instrument or other spectrophotometer device
- PacBio highly recommends using the Qubit High Sensitivity fluorometric assay for accurate dsDNA
quantitation
1.CHEF Mapper XA System (Bio-Rad)
2.Femto Pulse System (Agilent)
3.Pippin Pulse System (Sage Science)
A. DNA Sizing Characterization
http://www.sagescience.com/products/pippin-pulse/
http://www.bio-rad.com/en-us/category/pulsed-field-gel-electrophoresis-systems
https://www.agilent.com/en/product/automated-electrophoresis/femto-pulse-
systems
Recommended methods for determining gDNA size distribution:
Lane 1: 8-48 kb Ladder (Bio-Rad)
Lane 2: 5 kb Ladder (Bio-Rad)
Lane 3: HMW gDNA
Lane 4: Degraded gDNA
Lane 1: High MW gDNA
Lane 2: Degraded gDNA
Lane 3: 165 kb Ladder
Evaluation of gDNA quality using A) Bio-Rad CHEF
Mapper System and B) Femto Pulse System. Lanes 3 A and
1B are examples of high quality, high molecular weight
genomic DNA. Lanes 4A and 2B are examples of degraded
gDNA.
A B➢ Up to 10 Mb
➢ >16 Hour Run Time
➢ Up to 165 kb
➢ 1.5 Hour Run Time
➢ Up to 80 kb
➢ 16 Hour Run Time
Femto Pulse System only requires
~0.5 ng of gDNA sample and
assay run time is <1.5 hours
- DNA purity can be determined by using a NanoDrop® instrument or other spectrophotometers
- For ultrapure gDNA, A260/280 ratio is typically between ~1.8 - 2.0 and A260/230 ratio is ≥2.0
- If A260/280 and A260/230 readings are out of the range specified above, PacBio recommends
performing an AMPure® purification step followed by re-assessment of quantity and purity of
the gDNA sample
260/280 Ratio
▪ A low A260/A280 ratio may indicate the presence of protein, phenol, or other contaminants that absorb
strongly at or near 280 nm. Sometimes it may be caused by a very low concentration of nucleic acid.
▪ High 260/280 ratios are not indicative of an issue
260/230 Ratio
▪ A low A260/A230 ratio may be the result of:
❑ Carbohydrate carryover (often a problem with plants)
❑ Residual phenol from nucleic acid extraction
❑ Residual guanidine (often used in column-based kits)
❑ Glycogen used for precipitation
▪ A high A260/A230 ratio may be the result of:
❑ Making a blank measurement on a dirty pedestal of a Nanodrop instrument
❑ Using an inappropriate solution for the blank measurement
B. DNA Purity Determination
- Accurate quantitation of DNA concentration is critical for PacBio template preparation
procedures.
▪ Specifically, it is critical to determine the concentration of the double-stranded DNA, since only double-
stranded DNA will be converted into sequencing templates.
- PacBio highly recommends using a Qubit® fluorometer tool and Qubit ds DNA High
Sensitivity (HS) Assay Kit for routine DNA quantitation during SMRTbell library construction.
-When assessing gDNA QC, PacBio recommends using both fluorometric and
spectrophotometric methods – for example, using both the Qubit and NanoDrop instruments
▪ If the sample is pure gDNA, free of any RNA contaminants and other small molecules, the two methods
should converge to similar DNA concentration measurement values
C. DNA Quantification
- If the measured NanoDrop concentration is significantly different
(>50%) from the Qubit measurement, PacBio recommends doing an
AMPure purification step (as specified by your chosen library
preparation protocol), followed by a re-measurement with both
methods. Typically, a single AMPure purification step resolves the
discrepancy.
▪ If the concentration measurement discrepancy after one AMPure purification
step is not reduced, we recommend trying another cleanup approach before
a re-measurement with both methods.
SPECIAL HANDLING RECOMMENDATIONS FOR
PREPARING LARGE INSERT (>15 KB)
SMRTBELL LIBRARIES
1. For small numbers of samples, use DNA Lo-Bind 1.5 mL microcentrifuge tubes for all
enzymatic and AMPure PB bead purification steps.
2. Multi-channel pipettes and 96-well plates/PCR strip tubes may be used to efficiently
process large numbers of samples.
3. Use wide-bore tips for all pipette mixing steps when preparing >15 kb size-selected
libraries.
4. Never vortex tubes containing high-molecular weight DNA samples. Mix by gentle pipetting
unless stated otherwise.
5. Always follow best practices for DNA quantitation using a Qubit fluorometer system:
- Use the Qubit dsDNA High Sensitivity (HS) Assay reagent kit.
www.pacb.com
For Research Use Only. Not for use in diagnostic procedures. © Copyright 2020 by Pacific Biosciences of California, Inc. All rights reserved. Pacific Biosciences, the Pacific Biosciences logo, PacBio,
SMRT, SMRTbell, Iso-Seq, and Sequel are trademarks of Pacific Biosciences. Pacific Biosciences does not sell a kit for carrying out the overall No-Amp Targeted Sequencing method. Use of this
method may require rights to third-party owned intellectual property. BluePippin and SageELF are trademarks of Sage Science. NGS-go and NGSengine are trademarks of GenDx. FEMTO Pulse and
Fragment Analyzer are trademarks of Agilent Technologies Inc.
All other trademarks are the sole property of their respective owners.