Genomics, Epigenomics & Transcriptomics
Hasin et al. Multi-omics approaches to disease, Genome Biology 18:83 (2017).
Genomics, Epigenomics & Transcriptomics
DNA DNA & Histone RNA
Modifications
C. Petit. Evodevomics, BioSciences Master Reviews (2013).
Sequencing Library Preparation
Starting Material: DNA & RNA
RNA-Seq library prep
Genomic library prep
Mate-pair sequencing (circularization)
Low input / single cell library prep
High Quality Starting Material
DNA Extraction
Treat with RNase
RNA Extraction
Treat with DNase
Practice your extraction before the real experiment
Genomic DNA Extraction
https://genomics.ed.ac.uk/resources/sample-requirements
Any of the available kits or protocols
Bacteria or yeast often need additional bead-beating step.
RNase treat all DNA samples.
Quantitation
Absorbance: Nano-drop (50-500 ng/ul)
Theoretically should can read to 3000 ng/ul. Empirically find it is
only accurate within range above.
Dye based
SYBR Green, Qubit, Quant-IT
Genomic DNA QC
https://genomics.ed.ac.uk/resources/sample-requirements http://www.agilent.com/cs/library/applications/5991-5258EN.pdf
Agarose Gel
TapeStation
Genomic DNA Assay
Chromatin Immunoprecipitation (ChIP)
https://www.thermofisher.com/us/en/home/life-science/antibodies/antibodies-learning-center/antibodies-resource-library/antibody-application-notes/step-by-step-guide-successful-chip-assays.html
Crosslinking
Cell Lysis
Chromatin Preparation / Sheering
Immunoprecipitation
Crosslinking Reversal & DNA cleanup
RNA: What is Transcriptomics?
Wikipedia
“The transcriptome is the set of all messenger RNA molecules in one
cell or a population of cells.”
National Cancer Institute Definition of Terms
“The study of all RNA molecules in a cell. Transcriptomics is used to
learn more about how genes are turned on in different types of cells
and how this may help cause certain diseases, such as cancer.”
RNA enrichment
PolyA tailed messenger RNA: mRNA-Seq
Total RNA (rRNA removed): “total” RNA-Seq
Front Genet. 2015 Jan 26;6:2
mRNA (polyA) Purification
mRNA enrichment
mRNA binds beads coated with oligo dT primer
Non-polyadenylated transcripts are washed away
TTTTTT AAAA
AAAA
TTTTTTAAAA
Transcripts Lost in polyA Purification
Ribosomal/Transfer RNA
Histone mRNA
Long-noncoding RNA
Nascent intron containing transcripts
Micro RNA
Degraded RNA
Many viral transcripts
Prokaryote/Bacterial transcripts
polyA is the degradation signal
rRNA Depletion
Illumina: TruSeq
Probes hybridize rRNA on magnetic
beads
RNA of interest remains in supernatant
KAPA: RiboErase
Probes hybridize rRNA in solution
Hybrids are digested with RNase H
Probes digested with DNAse I
Modified from: Scientific Reports 6, article 37876 (2016)
Bead Rnase H
rRNA
Purified RNA
mRNA/long noncoding RNA/nascent RNA
rRNA
RNA Extraction
Cultured cells – Easy!
Tissue samples
Use your favorite column kit – Qiagen, Invitrogen, Zymo
For high throughput suggest bead based – MagMax
96 well format column plates are also available
Dissect in cold room if possible
Use RNAlater solution to store tissue sample
Upon extraction proceed to homogenization in cold room
RNA Extraction: Column Purification
On-column DNase digestion
Dry to remove excess ethanol
Elute with warm water to
increase yieldsOn-Column
DNase
http://www.sabiosciences.com/pathwaymagazine/pcrhandbook/10.php
RNA Extraction: Column Purification
Standard columns/protocols have 100-200bp cut off.
Will result in loss of small RNA species
Specialized columns and kits are available
microRNA
FFPE
Blood
HACK - Cut off size can be adjusted by changing the
percent ethanol used for sample binding
RNA Extraction: Bead Purification
Magnetic based purification good for high-throughput
applications
Can use oligo dT beads or total RNA beads
DNase step:
DNase I digestion step is sometimes skipped (polyA libraries only)
Best practice is to keep this step.
Binding Wash DNase Re-bind Wash Elute
http://www.beckman.com/nucleic-acid-sample-prep/rna-isolation/isolation-from-tissue
RNA Extraction: Tissues / Trizol
Keep tissues as cold as possible
Work in cold room
After homogenization suggest column based cleanup
Particularly important if used Trizol for lysis
DNase 10ug then column cleanup
https://www.thermofisher.com/order/catalog/product/AM7020
RNA Quantitation & Quality
Quantitation
Absorbance: Nano-drop (50-500 ng/ul)
Theoretically should can read to 3000 ng/ul. Empirically find it
is only accurate within range above.
Dye based
RiboGreen
Qubit / Quant-IT
Quality
Visualize on gel
Agilent Bioanalyzer (RIN)
RNA quality
High quality RNA needed for mRNA libraries
Degraded samples should only be used to make
a “total” RNA-seq library – rRNA removal
FFPE & Archival Samples
mRNA Purification of Degraded Samples
Transcript 1 Transcript 2
PolyA tail no longer attached to transcript.
Results in differential loss of transcripts between samples.
TTTTTT AAAA
AAAA
TTTTTTAAAA
AAAA
Sequencing Library Preparation
Starting Material: DNA & RNA
RNA-Seq library prep
Genomic library prep
Mate-pair sequencing (circularization)
Low input / single cell library prep
RNASeq Stranded Library Prep
(dUTP method)
Index
or strand specific amplification
or mRNA purification
http://www.rna-seqblog.com/wp-content/uploads/2012/12/library-preparation.jpg
Library Strandedness
http://seqanswers.com/forums/showthread.php?t=44220
ACCATGAACCGTA
TGGTACTTGGCAT
ACCAUGAACCGUA
Read alignment depends on
direction of transcription
“sense” strand of transcript can
be on either the sense or
antisense strand of the DNA
Library Strandedness
https://galaxyproject.org/tutorials/rb_rnaseq/
Key steps in library preparation
Starting Material
Library amplification bias
Multiplexing
qPCR quantitation
Sequencing read order & terminology
Library Amplification Bias
Final step of library prep is
amplification
Introduces library bias
Some products preferentially
amplified
Fewer cycles = less bias
Modified from: Nature Methods 9, 72-74 (2012)
transcript count
2, 13, 4
transcript count
12, 20, 8
Limited Cycle Library Amplification
Number of cycles needed is proportional to
amount of input RNA.
Library prep kits will recommend a certain
number of cycles.
This is usually optimized for the lower input.
Test how many cycles will give you
enough product.
Fewer cycles = less bias
Perform micro qPCR reaction on small amount of pre-
amplification library (Kapa)
Amplify only the number cycles needed to get enough
product for sequencing (20ul of 4nM product)
Limited Cycle Library Amplification
https://www.kapabiosystems.com/document/kapa-library-quantification-illumina-tds/?dl=1
Limited Cycle Library Amplification
9 cycles | 10 cycles 11 cycles | 12 cycles
Amplify 9 cycles
PCR
bubble
Library QC
Quantitation
Dye based
SYBR Green
Qubit / Quant-IT
Size & Quality
Agilent Bioanalyzer
Size determination
Do not use for
quantitation
Peak around 150 = primer dimer
Size selection with SPRI beads
http://core-genomics.blogspot.com/2012/04/how-do-spri-beads-work.html
Solid Phase Reverse
Immobilization beads
Carboxyl groups on
surface bind DNA in the
presence of crowding
agents (PEG & NaCl)
Key steps in library preparation
Starting Material
Library amplification bias
Multiplexing
qPCR quantitation
Sequencing read order & terminology
RNASeq Stranded Library Prep
(dUTP method)
Index
or strand specific amplification
http://www.rna-seqblog.com/wp-content/uploads/2012/12/library-preparation.jpg
or mRNA purification
Multiplexing (barcodes and indices)
Multiplexing allows optimal use of reads you will get
Charges for sequencing are usually per lane of the flow cell
For RNA-Seq number of reads you need will depend on your
experiment
HiSeq generates ~150 million reads per lane
NextSeq generates ~ 450 million reads (one lane instrument)
10 million standard for transcriptome
20 million standard for total RNA (rRNA depleted)
Make sure multiplexing libraries of similar size
Consider Cluster Size in Multiplexing
Library Preparation
DNA
(0.1-5.0 μg)
1 2 3 7 8 94 5 6T G CT A C G A T …
C
C
CC
A
A
A
TT
GG
G
G
Sequencing
Single molecule array
Cluster Growth
Image Acquisition Base Calling
5’
5’3’
TG
TA
CG
AT
CA
CC
CG
AT
CG
AA
www.support.illumina.com
Multiplexing
Pool samples based on dye based quantitation
Submit pool to core facility for sequencing.
Make all sequencing libraries in one batch
qPCR quantitate
before sequencing
Key steps in library preparation
Starting Material
Library amplification bias
Multiplexing
qPCR quantitation
Sequencing read order & terminology
Sequencing Read Order
1. Read 1
2. Index Read 1 (i7)
3. Index Read 2 (i5)
4. Read 2
HiSeq/MiSeq (4 color)
• A&C read on one camera
• G&T read on other
NextSeq (2 color)
Barcode and/or UMI
INDEX
Rd2 Seq PrimerIndex 2
primer(A)
Index 2
primer(B)
Index 1 primerRd1 Seq Primer
Sequencing Library Preparation
Starting Material: DNA & RNA
RNA-Seq library prep
Genomic library prep
Mate-pair sequencing (circularization)
Low input / single cell library prep
Genomic Library Prep
Once you have sheared your DNA this is a quick process
Protocol same as for RNA-Seq once
you have sheared dsDNA
Acoustic shearing – Covaris
Sonication
Hydrodynamic shearing – nebulization
Shear Genomic DNA or begin with cDNA
End Repair (blunt ends)
Add 3’ A Tail
Ligate Adapters
Enrich/Linearize with PCR
Sequencing
http://tucf-genomics.tufts.edu/home/faq
Tagmentation(DNA fragmentation facilitated by transposon activity)
http://www.molecularecologist.com/2015/01/new-to-the-genome-sequencing-8-menu-nextera-library-preps/
Tagmentation Approach
Nextera from Illumina
Very fast and efficient for DNA library preps
Often used as last stage of low input RNASeq library protocols
Works with small amounts of DNA
Important to RNase treat your sample
Needs precise DNA quantitation (Qubit)
https://www.my46.org/intro/whole-genome-and-exome-sequencing
Whole Genome Sequencing (WGS)
vs
Whole Exome Sequencing (WES)
Genomic Assembly with Mate-Pairs
https://www.illumina.com/content/dam/illumina-marketing/images/technology/mate-pair-sequencing-figure.gif
http://www.illumina.com/
Read2
Read1
Read2
Read1
Go
ing
b
ac
kw
ard
s
Mate-Pair Sequencing
http://www.illumina.com/
Read2
Read1
Read2
Read1
Read1
Read2
Go
ing
b
ac
kw
ard
s
Mate-Pair Sequencing
Insert pieces of DNA can be long (2-5kb)
Allows for better de novo genome assembly
and finishing of genome assemblies.
Helps in determining presence and location of
genomic rearrangements and amplifications.
Mate-Pair Genomic Library
Sequencing Library Preparation
Starting Material: DNA & RNA
RNA-Seq library prep
Genomic library prep
Mate-pair sequencing (circularization)
Low input / single cell library prep
Low Input & Single Cell RNA-Seq
Lower input = less chance to see mRNA of interest
Need to consider sampling error
High technical variation
Single cell methods will only capture 10-40% of
expected mRNA
Will not reliably detect low-abundance transcripts
Differential expression observed is reliable for highly
expressed genes
Smart-Seq / Drop-Seq / SCRB-Seq
S. Picelli et al., Full-length RNA-seq from single cells using Smart-seq2., Nat Protoc 9, 171–81 (2014).
In Drop-Seq / SCRB-Seq
libraries are enriched for 3’
UMI labeled ends
InDrops Single Cell Sequencing
Lysis and reverse transcription occurs in the beads
Samples are frozen after RT as RNA:DNA hybrid in gel.
A. M. Klein et al., Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells., Cell 161, 1187–201 (2015).
InDrops Library Prep / CEL-Seq2
ssSynthesis to make full
length dsDNA
IVT back to RNA via T7
promoter
Fragmentation RNA
Random hexamer RT
with adaptor
PCR to add index and
illumina adaptorsR. Zilionis et al., Single-cell barcoding and sequencing using droplet microfluidics, Nature Protocols 12, 44-73 (2017).
InDrops
Smart-Seq gives full transcript information &
detects most genes per cell.
Droplet based and Smart-Seq most accurate
differential expression
Drop-Seq 5-12% capture efficiency
InDrops/10x 50-80% capture efficiency
Single Cell / Low Input Methods
Low Input or Single Cell
Only use if needed for experiment
All commonly used methods all rely on PolyA tail
If you can get more starting material then you will
get better results.
Gold standard is TruSeq with >500ng input RNA
Plan a small scale starter experiment to see if
protocol will give useful results
http://rnaseq.uoregon.edu/
Transcript Enrichment:
Capture Sequencing
Capture targeted sequence using
biotinylated RNA bait
Sequencing library applied to
beads
Retain only library covering genes
of interest
Saves money on sequencing
IDT lockdown probes expensive
but good for small number genes
http://rnaseq.uoregon.edu/
Capture Probes
Tiling is the number of times a base is covered by a different
probe.
Difficult to design probes if looking at a single gene family or
pseudogenes.
Final Thoughts
Practice your library prep on a control sample.
Be sure you understand each step in library prep.
Talk to someone who has done the protocol before
starting.
qPCRPrecise quantitation is key to effective sequencing!