2 nd Genera-on ( NextGen ) Sequencing Technologies

Transcription

1 2 nd Genera-on ( NextGen ) Sequencing Technologies Jay Shendure Read Length is Not As Important For Resequencing % of Paired K-mers with Uniquely Assignable Location 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Length of K-mer Reads (bp) ECOLI HUMAN Paired End Reads are Important! Known Distance Read 1 Read 2 Repe--ve DNA Unique DNA Paired read maps uniquely Single read maps to mul-ple posi-ons 1

2 Illumina ~ 400 bp 2

3 Each piece has a unique sequence 3

4 bridge PCR 4

5 thousands of strands/cluster each cluster ( polony ) has a unique sequence 5

6 OH Metzger M (2009) Nature Reviews Gene-cs 11: OH 6

7 100+ Million Clusters Per Flow Cell Illumina, EEST, WUSTL GCTGA 100 Microns 7

8 Camera & computer -me is limi-ng! Chemistry problem 1: terminator is retained Flowcell 8 lanes out of phase For picture taking: Each lane is broken up into 100 -les, each fluor is imaged separately 2400 pictures taken per cycle Chemistry problem 2: fluor is retained 1000 Gb/run $50,000 Read length bp 330 human genomes HiSeq human genomes at 30X 8

9 Ion Torrent emulsion PCR empcr Margulies M et al, (2006) Genome sequencing in microfabricated high- density picolitre reactors Nature 437, Nature 475:348 (2011) ~100 bp reads 30 Mb/run 9

10 PMID: million SNPs 454 technology, 74X, 245 Gb cost < $1M 10,654 cause aa subs-tu-on (7,648 different from Venter) 222,718 indels (2 to 40kb) 18 CNVs (26 kb to 16 Mb) carrier of 10 highly penetrant disease alleles Illumina, 73X, 173 Gb con-g N50 = 40 kb scaffold N50 = 13 Mb 30 volume 42 number 1 january 2010 GENETICS : a recessive EMS- induced muta-on affec-ng egg shell morphology Illumina, 8X coverage 103 SNP differences between mutant and wt 9 non- synonomous 2 nonsense >> one in encore, an obvious candidate Illumina 51 Gb of sequence 76 bp reads 40X coverage 4 affected individuals 10

11 11 Pepke S, Wold B & Mortazavi A (2009) Nature Methods 6:S22 RNA- Seq ChIP- Seq Lefrançois P et al (2009) Efficient yeast ChIP- Seq using mul-plex short- read DNA sequencing BMC Genomics 10:37 3 rd Genera-on Sequencing Technologies A T G C A T G C A T G C A T G C A T G C A T G C A T G C

12 Single- molecule sequencing Gupta PK (2008) Single- molecule DNA sequencing technologies for future genomics research Trends Biotechnol 26: Nanopore sequencing - Nanopore sequencing + Gupta PK (2008) Single- molecule DNA sequencing technologies for future genomics research Trends Biotechnol 26: Gupta PK (2008) Single- molecule DNA sequencing technologies for future genomics research Trends Biotechnol 26:

13 Pacific Biosciences Single- molecule sequencing emission excita-on emission ZMW: a hole, tens of nanometers in diameter, fabricated in a 100nm metal film deposited on a silicon dioxide substrate detec-on volume 20 zeptoliters (10-21 liters) Eid et al 2008 PacBio technology backgrounder: hwp://wwwpacificbiosciencescom/indexphp?q=technology- introduc-on When the DNA polymerase encounters the nucleo-de complementary to the next base in the template, it is incorporated into the growing DNA chain During incorpora-on, the enzyme holds the nucleo-de in the ZMWs detec-on volume for tens of milliseconds, orders of magnitude longer than the average diffusing nucleo-de The system detects this as a flash of bright light because the background is very low The polymerase advances to the next base and the process con-nues to repeat PacBio technology backgrounder: hwp://wwwpacificbiosciencescom/indexphp?q=technology- introduc-on PacBio technology backgrounder: hwp://wwwpacificbiosciencescom/indexphp?q=technology- introduc-on 13

14 Eid J et al (2009) Molecules Real- Time DNA Sequencing from Single Polymerase Molecules Science 323, 133 PMID: F Sanger, S Nicklen, and A R Coulson, Proc Natl Acad Sci U S A 1977; 74: