Next Generation Sequencing-based approaches

Epigenetic Epidemiology Short Course

Session 5 - Assay design and sample processing Wednesday 13th June 2012

Dr Jonathan Coxhead

NIHR Biomedical Research Centre Institute of Genetic Medicine

Newcastle University

Overview

• What is next generation sequencing (NGS)? • Basic concepts • Current Platforms • Targeting your genomic region of interest • NGS methods specific to epigenetic studies • Where should I get my sequencing done? • Budget considerations • Sample submission • Bioinformatics and IT • NGS bioinformatics tools and epigenetics • Bench top sequencing • Future trends – single molecule sequencing

What is next generation sequencing?

Jonathan Rothberg 454

“Since 1986, Jonathan Rothberg says he has wanted to improve ease and cost of gene sequencing. In 1999, when his son was born, Rothberg spent two weeks on paternity leave and made arguably his biggest leaps toward this goal.” Nature Methods - 5, 11 - 14 (2008) doi:10.1038/nmeth1154

↑↑↑ capacity ↑ speed

(comparative scale)

↓ cost (comparative

scale)

ER Mardis. Nature 470, 198-203 (2011) doi:10.1038/nature09796

Changes in instrument capacity over the past decade, and the

timing of major sequencing projects

Methodology – variations on a theme

• DNA fragmentation

• Adaptor ligation (include barcode sequence)

• Immobilise to a solid surface

• Clonal amplification

• Step-wise order of sequencing

• Photo detection (not always)

• Fragment DNA

• Adaptor ligation

• Bead Capture

• emPCR

• Emulsion breaking

• Sequencing

Roche GS-FLX (454)

Illumina

• Fragment DNA

• Adaptor ligation

• Cluster formation

• Sequencing

SOLiD (Life Technologies)

•DNA fragmented and

adaptors added

•emPCR similar to 454 but

all automated

•Beads covalently bound to

a glass slide

•Sequencing by

Oligonucleotide Ligation

and Detection

Ion Torrent (Life Technologies)

• Preparation workflow

similar to the 454 but

all automated

• Semi-conductor

sequencing

Whole genomes to single exons

• Whole genome DNA

• Whole exome captured DNA (Agilent, NimbleGen, Illumina)

• Custom capture DNA (Agilent and NimbleGen)

• PCR products and LR-PCR products targeting exons

• Paired end, transcriptome, metagenomic, plasmid, CNV

• ChIP Sequencing

• Methylation

• Small RNA

Roche GS-FLX – Epigenetic options

• Detection and Quantification of Methylation Patterns using Amplicon Sequencing

• DNA isolation – QIAamp DNA Mini Kit (Qiagen) • Bisulphite modification – EZ DNA Methylation Kit (Zymo Research) • Optimal amplicon placement is crucial and should ideally be determined using

literature evidence of differential methylation. • Commonly look at a region 1000 bases upstream of the transcription factor

start site and 500 bases after the first exon • <j.m.coxhead@ncl.ac.uk>

Illumina – Epigenetic options

• ChIP Sequencing using the ChIP-Seq DNA Sample Prep Kit

• 10 ng ChIP enriched DNA

• Read length of 25 – 32 nt sufficient for mapping

• 1 sample per 8 lane flow cell Whole-Genome Chromatin IP Sequencing (ChIP-Seq) http://www.illumina.com/documents/products/datasheets/datasheet_chip_sequence.pdf

ChIP-Seq Data Analysis http://www.illumina.com/Documents/products/technotes/technote_chip_seq.pdf

• Methylation Analysis

• Whole-Genome Bisulphite Sequencing using the Paired-end DNA Sample Prep Kit

• 5 µg genomic DNA Whole Genome Bisulphite Sequencing for Methylation Analysis https://icom.illumina.com/download/summary/3xkQCsK7KUane-IlxkVdXg

• Reduced Representation Bisulphite Sequencing using the Paired-end DNA Sample Prep Kit

• 5 µg genomic DNA RRBS for Methylation Analysis https://icom.illumina.com/download/summary/aOaPMUxJz0ae9IHZZoA8dg

• Small RNA Sequencing using the TruSeq RNA Sample Prep Kit

• 1 ug high quality total RNA TruSeq Small RNA Sample Prep Guide https://icom.illumina.com/download/summary/ATZRuMiBPkukcRQOJ792Xg

SOLiD – Epigenetic options

• ChIP-Seq using the MAGnify ChIP Kit

– Low sample input 167 µg fresh frozen tissue

– 10 000 primary cells

– 1 – 10 ng ChIP enriched DNA

– Recommend 10 M reads for ChiP Sequencing Analysis of Protein-DNA Interactions With the SOLiD ChiP-Seq Kit http://tools.invitrogen.com/content/sfs/brochures/cms_081785.pdf

• Methylation Analysis

– Enzymatic Genome Partitioning

– Enriching for Methylated DNA (LifeTech recommend the MethylMiner Methylated DNA Enrichment Kit)

– Bisulphite Conversion (LifeTech recommend the Cells-to-CpG Bisulphite Conversion Kit - can be used with FFPE samples)

Options chosen will depend on the target genome size and the level of resolution required – consult literature (60 M reads per sample?)

• Small RNA and miRNA Sequencing using the Total RNA-Seq Kit

– From 5 ng miRNA starting material

– Detect changes in in molecules present at <1 copy per cell

– Analyse FFPE samples

– Recommend 10 M mappable reads for profiling known miRNAs in humans Whole Genome Analysis of Small RNA http://tools.invitrogen.com/content/sfs/brochures/cms_057582.pdf

miRNA Discovery and Profiling with the SOLiD Small RNA Expression Kit http://tools.invitrogen.com/content/sfs/brochures/cms_057560.pdf

FFPE Small RNA Library Preparation for SOLiD Sequencing http://tools.invitrogen.com/content/sfs/manuals/cms_092090.pdf

Ion Torrent – Epigenetic options

• ChIP Sequencing using the MAGnify Chromatin Immunoprecipitation System Fast and Cost Effective ChIP-Sequencing with PGM System:ChIP-Seq on a Chip http://lifetech-it.hosted.jivesoftware.com/docs/DOC-2623

• ChIP Sequencing at the Broad Institute Broad Institute - Ion Torrent Applications Development: Evaluation of ChIP-Seq on Ion Torrent http://lifetech-it.hosted.jivesoftware.com/docs/DOC-2221

• ChIP Sequencing using the Diagenode iDeal ChIP-Seq Kit PGM ChIP-Seq using Diagenode’s iDeal ChIP-Seq Kit http://lifetech-it.hosted.jivesoftware.com/docs/DOC-2670

• Methylation Analysis

• NB – couldn’t find any information regarding the sequencing of bisulphite modified DNA on the Ion Community website. I would have no hesitation in trialling this using an amplicon sequencing approach, and imagine there are groups doing this already.

• Small RNA Sequencing using the Ion Total RNA-Seq Kit

• 5 ng miRNA

• 35 – 200 nt read length for sequencing miRNA Ion RNA-Seq Technical Note (Small RNA) http://www.iontorrent.com/lib/images/PDFs/technical%20note_ion%20rna-seq%20solution_co23050.pdf

Ion Total RNA-Seq Kit for Small RNA Libraries http://lifetech-it.hosted.jivesoftware.com/docs/DOC-1476

Outsourcing sequencing – consumer guide

• Seqanswers (The Next Generation Sequencing Community) http://seqanswers.com/

• Next Generation Genomics: World Map of High-throughput Sequencers

http://omicsmaps.com/

• Q’s – How commercial?

– Previous experience - case studies & publications?

– Turn around times?

– Customer care – communication, advice, alternative strategies?

– Data delivery – what and how?

– £$€?

• Outsource the decision making e.g. BlueSeq (the Global Sequencing Service Exchange) http://www.blueseq.com/

• No decision to be made?

Budget considerations

• Don’t blow all your £$€ on the sequencing

• £$€ sample prep – DNA extraction etc?

• £$€ higher performance PC?

• £$€ software for data analysis?

• £$€ data storage – need to use a cluster?

• £$€ bioinformatics support?

• £$€ for follow up validation (could this cost more that the sequencing?)

Providing good starting material for sequencing

• It’s entirely your responsibility to provide the best quality starting material that you can

• Nano-drop - assess sample quality (260/280)

• Lab on a Chip (Agilent) - assess sample quality

• PicoGreen (Invitrogen) - sample concentration

• Provide gel photo of fragment against an appropriately sized ladder.

Bioinformatics and IT

Bioinformatics

done by …..

Pros? Cons?

Service

provider

Quick and

reproducible?

Black box

Local

Bioinformatician

Flexibility –

custom pipeline

Scale of work?

Maintenance?

Commercial

Software

Best of, up-dates

& tech support

Single solution and

expensive, unused

features?

Independently with

open source tools Flexibility &

knowledge gain

Skill set and

time available?

Analysis software

• Commercial Software

NextGENe (Softgenetics) http://www.softgenetics.com/NextGENe.html

CLCbio Genomics Workbench http://www.clcbio.com/index.php?id=1240

GenomeQuest https://www.genomequest.com/

DNAStar http://www.dnastar.com/

• Web based applications – reproducible workflows

Galaxy http://galaxy.psu.edu/

Taverna http://www.taverna.org.uk/

• Free Software

Seqanswers

http://seqanswers.com/wiki/Software

Data storage

• Make IT staff your new BFFs

Storage I’ve used

• Decent sized hard drive / cluster (analysis)

• Tape (weekly + fireproof safe)

• Rack mounted NAS (daily)

• Virtual machine / cloud (file sharing)

Bench top sequencing

Roche GS-Junior Ion Torrent PGM Illumina MiSeq

NGS – next 5 yrs

• Single molecule sequencing

– PacBio (now)

– Oxford Nanopore (next)

• $1000 genome 2012?

• Portable / handheld devices