Surya Saha Cornell University & Boyce Thompson Institute

suryasaha@cornell.edu // Twitter:@SahaSurya

IIT Indore

May 29, 2014

Slides: http://bit.ly/IITIndoreSeq

http://www.acgt.me/blog/2014/3/7/next-generation-sequencing-must-die

5/29/2014 IIT Indore 2

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53

DNA Structure discovery

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77

20

12

Sanger DNA sequencing by chain-terminating inhibitors

19

84

Epstein-Barr virus

(170 Kb)

19

87

Abi370

Sequencer

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95

20

01

Homo sapiens (3.0 Gb)

20

05

454

Solexa

Solid

20

07

20

11

Ion Torrent

PacBio

Haemophilus influenzae (1.83 Mb)

20

13

Slide credit: Aureliano Bombarely

Sequencing over the Ages

Illumina

Illumina Hiseq X

454

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Pinus taeda

(24 Gb)

20

14

MinION

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Its all about the $£€¥

http://www.genome.gov/sequencingcosts/

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First generation sequencing

Sanger method

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Frederick Sanger 13 Aug 1918 – 19 Nov 2013 Won the Nobel Prize for Chemistry in 1958 and 1980. Published the dideoxy chain termination method or “Sanger method” in 1977

http://dailym.ai/1f1XeTB

Sanger method

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http://bit.ly/1g6Cudq

http://bit.ly/1lcQO4J

Maxam-Gilbert method

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Maxam-Gilbert method

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http://bit.ly/1noY0fu http://bit.ly/1lGvJCA

First generation sequencing

• Very high quality sequences (99.999%)

• Very low throughput

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Run Time Read Length Reads / Run

Total

nucleotides

sequenced

Cost / MB

Capillary

Sequencing

(ABI3730xl)

20m-3h 400-900 bp 96 or 386 1.9-84 Kb $2400

http://bit.ly/1clLps3 http://1.usa.gov/1cLqIRd

Next generation sequencing

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http://bit.ly/1keDtZQ

• Second generation • Third generation • Fourth generation • Next-next-generation • Next-next-next

generation http://www.acgt.me/blog/2014/3/10/next-generation-sequencing-must-diepart-2

Use the specific technology used to generate the data

– Illumina Hiseq/Miseq/NextSeq

– Pacific Biosciences RS I/RS II

– Ion Torrent Proton/PGM

– SOLiD

– 454

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http://www.acgt.me/blog/2014/3/10/next-generation-sequencing-must-diepart-2

454 Pyrosequencing

One purified DNA fragment, to one bead, to one read.

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http://bit.ly/1ehwxWN

GS FLX Titanium

http://bit.ly/1ehAcEh

Illumina

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Output 15 Gb 120 GB 1000 GB 1800 GB

Number of Reads

25 Million 400 Million 4 Billion 6 Billion

Read Length

2x300 bp 2x150 bp 2x125 bp (2x250 update mid-2014)

2x150 bp

Cost $99K $250K $740K $10M

Source: Illumina

Illumina

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Output 15 Gb 120 GB 1000 GB 1800 GB

Number of Reads

25 Million 400 Million 4 Billion 6 Billion

Read Length

2x300 bp 2x150 bp 2x125 bp (2x250 update mid-2014)

2x150 bp

Cost $99K $250K $740K $10M

Source: Illumina

$1000 human genome??

Illu

min

a

5/29/2014 IIT Indore 17 http://1.usa.gov/1fP9ybl

Illu

min

a: M

ole

culo

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http://bit.ly/1aEPOBn

Pacific Biosciences SMRT sequencing

Single Molecule Real Time sequencing

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http://bit.ly/1naxgTe

Pacific Biosciences SMRT sequencing Error correction methods

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Hierarchical genome-assembly process (HGAP)

PB

Jelly

Enlish et al., PLOS One. 2012

PBJelly

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Pacific Biosciences SMRT sequencing Read Lengths

http://www.igs.umaryland.edu/labs/grc/

Mean Read Length: 8391 bp Maximum Subread Length: 24585 bp

Oxford Nanopore

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https://www.nanoporetech.com/

• No data yet

• Error model

http://erlichya.tumblr.com/post/66376172948/hands-on-experience-with-oxford-nanopore-minion

Others

• Ion Torrent Proton/PGM

• Nabsys

• SOLiD

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Comparison

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Next generation sequencing

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Run Time Read Length Quality

Total

nucleotides

sequenced

Cost /MB

454

Pyrosequencing 24h 700 bp Q20-Q30 0.7 GB $10

Illumina Miseq 27h 2x250bp > Q30 15 GB $0.15

Illumina Hiseq

2500 11days 2x125bp >Q30 1000 GB $0.05

Ion torrent 2h 400bp >Q20 50MB-1GB $1

Pacific

Biosciences 2h 5.5-8.5kb

>Q30 consensus

>Q10 single

400-800MB

/SMRT cell $0.33-$1

http://bit.ly/1clLps3 http://1.usa.gov/1cLqIRd

http://omicsmaps.com/

Next Generation Genomics: World Map of High-throughput Sequencers

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http://bit.ly/18pfUId

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http://bit.ly/18pfUId

Real cost of Sequencing!!

Sboner, Genome Biology, 2011

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Library Types

Single end

Pair end (PE, 150-800 bp, Fwd:/1, Rev:/2)

Mate pair (MP, 2Kb to 20 Kb)

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F

F R

F R 454/Roche

F R Illumina

Illumina

Slide credit: Aureliano Bombarely

Implications of Choice of Library

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Consensus sequence

(Contig)

Reads

Scaffold

(or Supercontig)

Pair Read information

NNNNN

Pseudomolecule

(or ultracontig)

F

Genetic information (markers)

NNNNN NN

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Quality control: Encoding

http://bit.ly/N28yUd

Phred score of a base is: Qphred = -10 log10 (e)

where e is the estimated probability of a base being incorrect

Which technology to use??

• Microbial genomes

• Eukaryotic genomes

• Resequencing genomes

• RNAseq and other XXXseq methods

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http://bit.ly/1ko9Kgh

Looking into the Crystal ball

• Desktop sequencing

• Diagnostics in the clinic

• Large scale environmental sequencing of microbes

• But challenges remain..

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• International Society of Computational Biology (ISCB)

• ISCB SC RSG India

• > 1500 members

• Contact – rsg-india@googlegroups.com

– http://www.iscbsc.org/rsg/rsg-india

– https://groups.google.com/forum/#!forum/compbio_discussion

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• Collaborate with student organizations

• Organize workshops and journal clubs

• Attend international meetings

Position available at Solgenomics

Cassavabase project

Plant Breeding + Bioinformatician

● Familiar with breeding

● Programming in Perl, R, SQL, Hadoop

● Linux

● Africa

● Genius

http://www.cassavabase.org/forum/posts.pl?topic_id=9

Thank you!! Questions??

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