Introduction to second generation sequencing

[MIT]
Introduction to 2GS data analysis
Drink faster !
June 23, 2011

Production Informatics and Bioinformatics
June 23, 2011
Produce raw sequence reads
Basic Production
Informatics
Map to genome and generate raw genomic features (e.g. SNPs)
Advanced
Production Inform.
Analyze the data; Uncover the biological meaning
Bioinformatics
Research
Per one-flowcell project

First Generation: Sanger sequencing
Second Generation: amplified molecule sequencing
Third Generation: single molecule sequencing
Brief history of sequencing
June 23, 2011
*
*
* Discussion about category

What steps are involved in sequencing ?
June 23, 2011
sequencing by synthesis (SBS) technology
Fragmentation
Library generation
Amplification
Sequencing
Analysis
Illumina Marketing:
“3h 10 minutes wet-lab
30 minutes dry lab”

Illumina sequencing: Library + Amplification
June 23, 2011
“Illumina Sequencing Technology” booklet

Illumina Sequencing: Synthesis + Imaging
June 23, 2011
“Illumina Sequencing Technology” booklet

Output: 1.5 Terabyte of data
June 23, 2011
Inspired by anzska information booklet

Sequencer Output Conversion: Production Informatics
1.5 TB data : 6 billion clusters with 100 bp reads
= 600 billion data points
June 23, 2011
HiSeq
CASAVA
…
× read length
For HiSeq: images are converted to flat files (*.bcl or *.cif)
visualpharm.com
Maysoft

Multiplexing
6 billion reads:
750 million reads per lane
Currently 12-plex (soon 96-plex):
One run
June 23, 2011
Oliver Twardowski

Demultiplexing
June 23, 2011
CASAVA
…
…
× samples
× read length
visualpharm.com

CASAVA1.8.0 program call
June 23, 2011
configureBclToFastq.pl
--input-dir Data/Intensities/BaseCalls/
-output-dir Data/Unaligned
--sample-sheet SampleSheet.csv
--use-bases-mask y100,I6nn,Y100 >file.log 2>&1
cd Data/Unaligned
qsub -pe make 16 -jy -v $MYPATH –oqsub.out -cwd –N fastq -by
make -j 16
Runtime: ~ 6h

Fastq files
June 23, 2011
@HWI-ST301_0112:1:1:1169:2044#0/1
CCATAAGGCCACGTATTTTGCAAGCTATTTAACTGGCGGCGAT
+HWI-ST301_0112:1:1:1169:2044#0/1
dddcdd^dd`acacdacd`ecdedabdcdddcc```bTa
36 36 36 35 28 …
ASCII @ .. ~
DEC 64 .. 126
PHRED 0 .. 62
Phred scores are estimates only !
Cock PJ, Fields CJ, Goto N, Heuer ML, Rice PM. The Sanger FASTQ file format for sequences with quality scores, and the Solexa/Illumina FASTQ variants. Nucleic Acids Res. 2010 Apr;38(6):1767-71. PMID:20015970

Fastq – PHRED quality
Pathological
June 23, 2011

Fastq: Quality control
Base-pair quality score
Adapter contamination
Uneven Amplification
June 23, 2011

Three things to remember
Don’t be fooled by marketing
Fastqfiles are not directly usable
Basic-run QC can be made from fastq file
June 23, 2011
“All modern genomics projects are now bottlenecked at the stage of data analysis rather than data production”
Ewan Birney
European Bioinformatics Institute
Wellcome Trust
David S. Roos Bioinformatics--Trying to Swim in a Sea of Data;Science 16 February 2001: Vol. 291 no. 5507 pp. 1260-1261 DOI: 10.1126/science.291.5507.1260

Next Week:
June 23, 2011
Abstract: This session will focus on identifying SNPs from whole genome, exome capture or targeted resequencing data. The approaches of mapping, local realigment, recalibration, SNP calling, and SNP recalibration will be introduced and quality metrics discussed.

Walk-in-clinic
June 23, 2011

First Generation: Sanger sequencing
Second Generation: amplified molecule sequencing
Third Generation: single molecule sequencing
Brief history of sequencing
June 23, 2011
*
*
* Discussion about category

Helicos
true Single Molecule Sequencing(tSMS)™ technology
Sequencing by synthesis but much more sensitive so no amplification
June 23, 2011

Life Technology - Ion Torrent
Hydrogen Ion is released by the incorporation of a nucleotide, which is measured by a semiconductor
Depending on which nucleotide wash cycle the signal coincides
June 23, 2011

PacBio
Immobilized polymerase at the bottom of a well
Fluorescent nucleotides float around and if they are incorporated they are held still for tens of milliseconds, which is the signal that is recorded
No upper limit on the length
June 23, 2011
http://www.pacificbiosciences.com/smrt-biology/smrt-technology?page=4

Nanopore
Molecule is sucked through a poor and the change in the membrane charge due to the different nucleotides is recorded.
June 23, 2011
http://www.nanoporetech.com/sections/index/82

Introduction to second generation sequencing

by Denis Bauer on Jun 22, 2011

Accessibility

Categories

Tags

Upload Details

Usage Rights

Statistics

Introduction to second generation sequencing — Presentation Transcript