[I0D51A] Bioinformatics: High-Throughput AnalysisNext-generation sequencing. Part 1: Technologies

Prof Jan AertsFaculty of Engineering - ESAT/SCDjan.aerts@esat.kuleuven.be

TA: Alejandro Sifrim (alejandro.sifrim@esat.kuleuven.be)

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Announcements

May 27th (9am-noon): evaluation

open book

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Note to self...

Upload s_1_sequence.txt and s_2_sequence.txt to Galaxy first...

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Overview

• linux refresher (6/5)

• next-generation sequencing technologies and applications (6/5)

• sequence mapping (13/5)

• variant calling - SNPs (20/5)

• variant calling - structural variation (20/5)

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Linux Refresher...

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Next-generation sequencing technologies

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General principle

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Big data...

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First vs second generation sequencing

Shendure & Ji, 2008

Sanger sequencing (1st gen) 2nd/next gen sequencing

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Korbel et al, 2007

Paired-end sequencing

General approaches

• 2nd generation: clonally amplified single molecules

• Roche 454 pyrosequencing

• Illumina Genome Analyzer -> HiSeq: reversible terminator technology

• ABI SOLiD: ligation-based extension

• Next-next-generation/3rd generation: true single molecule

• Helicos: Heliscore

• Pacific Biosciences: SMRT

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Mardis, 2011

Steps

template preparation

sequencing and imaging

data analysis

genome enrichment

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A. Genome enrichment

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Sequencing costs

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What?

Only sequence relevant parts of the genome instead of whole genome, e.g.:

• specific Mb-scale regions known to be involved in particular disease (e.g. based on GWAS)

• specific candidate genes belonging to disease pathway

• exome (= all exons)

=> how to isolate these from non-target sequence? “pulldown”

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Pulldown: on-array

Turner et al, 2009

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Pulldown: in-solution

Turner et al, 2009

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Performance metrics

• fold-enrichment: ratio of abundance of target sequences post-enrichment vs pre-enrichment

• capture specificity: fraction of sequence reads that map to target

• uniformity: relative abundance of individual targets after enrichment

• completeness: fraction of target bases detectably captured

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B. Template preparation

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Problem: most imaging systems not designed to detect single fluorescent event => need amplified templates

Aim: to produce a representative, non-biased source of nucleic acid material from the genome under investigation => population of identical templates

Steps:

1. shear DNA

2. amplify templates

Options: emulsion PCR (emPCR) or solid phase amplification

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emulsion = mixture of two or more immiscible (unblendable) liquids; e.g. mayonnaise, vinaigrette

emPCR: thousands of microreactors/micro-eppendorfs

one bead + one DNA molecule per microreactor => PCR to 1000s of copies

Amplification by emulsion PCR

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Metzker et al, 2010

Williams et al, 2006

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Solid-phase amplification

Metzker et al, 2010

http://bit.ly/6JYIUz

http://www.youtube.com/watch?v=77r5p8IBwJk&NR=1

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C. Sequencing and imaging

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Sequencing and imaging

Technologies:

1. cyclic reversible termination

2. sequencing by ligation

3. pyrosequencing

4. real-time sequencing

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Cyclic reversible termination

DNA synthesis is terminated after adding single nucleotide

start/stop/start/stop/start/stop/...

Illumina: 4-colour

sequencing steps

Metzker et al, 2010

sequencing result

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Helicos: 1-colour

Metzker et al, 2010

sequencing steps

Metzker et al, 2010

sequencing result

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Sequencing by ligation

http://bit.ly/fPh22X

sequencing steps

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http://bit.ly/fPh22X

sequencing result

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Pyrosequencing

Metzker et al, 2010

Metzker et al, 2010

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Real-time sequencing

“ZMW” zero-mode waveguide

DNA polymerase

“strobe sequencing”

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Run time Gb/run

Roche 454

Illumina

SOLiD

Helicos

PacBio

8.5 hr 45

9 days 35

14 days 50

8 days 37

? ?

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• base quality drops along read

Sanger > SOLiD > Illumina > 454 > Helicos

(“dephasing” within clusters)

• base calling errors

Accuracy - base calling error

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Accuracy - homopolymer runs

Issue for Roche 454:

39% of errors are homopolymers

A5 motifs: 3.3% error rate

A8 motifs: 50% error rate

Reason: use signal intensity as a measure for homopolymer length

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Ronaghi, Genome Res 11:3-11 (2001)

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http://mammoth.psu.edu/labPhotos/imageOfFlowgram.jpg

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Is it 4? Is it 5? Is it 4?

http://mammoth.psu.edu/labPhotos/imageOfFlowgram.jpg

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Consensus accuracy

Increase accuracy for SNP calling by increasing coverage:

Illumina: 20X

SOLiD: 12X

454: 7.4X

Sanger: 3X

Factors: raw accuracy + read length

How deep do you have to sequence? => Poisson distribution: “If you sequence at average of 10X, how much of the genome will be covered at least 5X”?

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Bentley et al, Nature 456:53-56 (2008)

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FASTQ file format

“@” + identifier

sequence

“+” + identifier (optional)

phred-based quality scores

phred quality score encoding

Wikipedia

example fastq entries (n=2)

example fasta entries (n=2)

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Sequence quality control

Is this good sequence? (essential!)

E.g.: using FastQC tool (Babraham Institute, UK; http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/)

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Sequence quality control

per base sequence quality

good bad

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Sequence quality control

per sequence quality scores

good bad

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Sequence quality control

per base sequence content

good bad

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Sequence quality control

per base GC content

good bad

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Sequence quality control

per sequence GC content

good bad

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Sequence quality control

k-mer content

good bad

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Intermezzo: Galaxy

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Online genome analysis

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http://galaxy.psu.edu/

“Galaxy allows you to do analyses you cannot do anywhere else without the need to install or download anything. You can analyze multiple alignments, compare genomic annotations, profile metagenomic samples and much much more...”

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Applications of next-generation sequencing

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Kahvejian et al, 2008

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Kahvejian et al, 2008

DNA-seq

ChIP-seq

RNA-seq

575150

Kahvejian et al, 2008

DNA-seq

ChIP-seq

RNA-seq

identify sequence variations

identify pathogens

Exercises

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Try to login to the server mentioned on Toledo with username and password provided there.

There are 2 FASTQ files in /mnt/homes/jaerts/: s_1_sequence.txt and s_2_sequence.txt (= paired ends)

• How many sequences are in s_1_sequence.txt?

• What encoding was used for the quality score? Illumina? Sanger?

• What are the numerical quality scores for the first sequence in s_1_sequence.txt (i.e. 7172283/1)?

• Create an account on the Galaxy server

• Download s_1_sequence.txt and s_2_sequence.txt from Toledo and upload them into Galaxy. These files are also available on the linux server

• Have a look at the contents of s_1_sequence.txt.

• Convert quality scores to numeric values for s_1_sequence.txt (“FASTQ Groomer”)

• Draw the quality score boxplot for s_1_sequence.txt

• Draw the nucleotide distribution chart for s_1_sequence.txt

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Bentley DR et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456: 53-59 (2008)

Kahvejian A, Quackenbush J & Thompson JF. What would you do if you could sequence everything? Nature Biotechnology 26: 1125-1133 (2008)

Korbel JO et al. Paired-end mapping reveals extensive structural variation in the human genome. Science 318: 420-426 (2007)

Mardis ER. A decade’s perspective on DNA sequencing technology. Nature 470: 198-203 (2011)

Metzker ML. Sequencing technologies - the next generation. Nature Reviews Genetics 11:31-46 (2010)

Shendure J & Ji H. Next-generation DNA sequencing. Nature Biotechnology 26:1135-1145 (2008)

Turner EH et al. Methods for genomic partitioning. Annual Review of Genomics and Human Genetics 10 (2009)

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References