Find more extraction protocol recommendations for your sample type, plus guidance on DNA storage and contaminants: community.nanoporetech.com/extraction_methods

Find out more about size selection methods for long-read sequencing:

community.nanoporetech.com/extraction_methods

EXTRACTION: obtaining high molecular weight DNA

LIBRARY PREPARATION: selecting a kit

Resolving structural variants with long-read nanopore sequencingStructural variants (SVs), defined as variants spanning 50 bp or more, account for ten times as many variant bases as single nucleotide polymorphisms (SNPs) in the human genome1. With known causative effects in an extensive range of both normal and aberrant phenotypes, the need to comprehensively characterise SVs is becoming increasingly clear. With Oxford Nanopore, long-read sequencing of native DNA greatly improves the accuracy of detection of even the largest of SVs, including those regions inaccessible to other technologies2.

Here, we present a simple workflow for an effective whole-genome SV survey from a human blood sample, using the PromethION™ sequencing device.

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QIAGEN Gentra Puregene

Circulomics

QIAamp DNA Blood Midi KitQIAGEN Genomic-tip

60 min

High molecular weight gDNA

Optional fragmentationor size selection

End-prep and nick repair

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Ligation of sequencingadapters T

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Selecting a suitable extraction method is often a trade-off between input requirements, expected fragment lengths, lab experience and hands-on time. To maximise the volume of data at long read lengths, we recommend the QIAGEN Gentra Puregene Blood Kit.

There is no upper read length limit in nanopore sequencing, with reads routinely spanning tens or hundreds of kilobases and the current record spanning over 2 megabases. Fragmentation is optional: unfragmented DNA offers a simple workflow, but shearing and size selection can improve read N50. Internal testing has yielded good results from the Circulomics Short Read Eliminator Kit for size selection, and the Diagenode Megaruptor 3 for shearing.

To prepare gDNA for sequencing, we recommend the Ligation Sequencing Kit (SQK-LSK109), providing the greatest throughput and control over read lengths. Gentle pipetting with wide-bore tips can also help preserve long DNA strands.

Find out more about sample prep, including rapid and multiplexing options: nanoporetech.com/products/kits

WORKFLOW: STRUCTURAL VARIATION

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References:1. E. E. Eichler, 2019. NEJM. DOI: 10.1056/NEJMra18093152. M. T. W. Ebbert et al. 2019. Genome Biol. DOI: 10.1186/s13059-019-1707-2

Oxford Nanopore Technologies, the wheel icon, EPI2ME and PromethION are registered trademarks of Oxford Nanopore Technologies in various countries. All other brands and names contained are the property of their respective owners. © 2019 Oxford Nanopore Technologies.

Oxford Nanopore Technologies products are currently for research use only. WF_1042(EN)_V1_01Oct2019

Twitter: @nanoporewww.nanoporetech.com

For a human whole-genome survey, generating sufficient data for ~30x depth of coverage is a good starting point; this can be obtained by sequencing on a single PromethION Flow Cell for 72 hrs. Throughput is maximised by a nuclease flush and addition of fresh library every 24 hrs. For best metrics, we recommend upping this depth to >45x, or for a more cost-effective, light-touch approach, this could be lowered to 15x.

The PromethION is available configured for 24 or 48 individually-addressable flow cells, leaving the rest of the device free for other experiments to be started and stopped as needed. Basecalling can be carried out in real-time on the device, making use of the powerful onboard compute.

*Circulomics Short Read Eliminator Kit + Diagenode Megaruptor 3

Find out more about PromethION: nanoporetech.com/products/promethion

Find out more about data analysis solutions: nanoporetech.com/nanopore-sequencing-data-analysis

SEQUENCING: generating high yields on the PromethION

ANALYSIS: calling SVs without command line

To call variants in your long-read data, we recommend pipeline-structural-variation, available on github. The pipeline takes the FASTQ files produced by onboard basecalling, aligns to a provided FASTA reference genome, calls insertions, deletions and duplications >50 bp and outputs a VCF file and QC report. Our SV pipeline tutorial provides step-by-step instructions for the full process, including visualisation. View the tutorial here: community.nanoporetech.com/knowledge/bioinformatics

For those wishing to avoid command line, our cloud-based EPI2ME™ SV workflow provides fully automated human whole-genome SV analysis based on the same pipeline, generating a VCF file.

Find out more about nanopore sequencing service providers: nanoporetech.com/services/providers

Find out more at: nanoporetech.com/applications/structural-variation

FASTQ input file

VCF output file

QC & filter reads

Align reads minimap2

Call variants sniffles

Filter variants

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unfragmented

g-TUBE

SRE + MRIII*

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