Talk given at IIT Indore, India on May 29, 2014

1. 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

2. 5/29/2014 IIT Indore 2
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3. 1953
DNA Structure
discovery
1977
2012
Sanger DNA sequencing by
chain-terminating inhibitors
1984
Epstein-Barr
virus
(170 Kb)
1987Abi370
Sequencer
1995
2001
Homo
sapiens
(3.0 Gb)
2005
454
Solexa
Solid
2007
2011
Ion
Torrent
PacBio
Haemophilus
influenzae
(1.83 Mb)
2013
Slide credit: Aureliano Bombarely
Sequencing over the Ages
Illumina
Illumina
Hiseq X
454
5/29/2014 IIT Indore 3
Pinus
taeda
(24 Gb)
2014
MinION

4. 5/29/2014 IIT Indore 4
Its all about the $£€¥
http://www.genome.gov/sequencingcosts/

5. 5/29/2014 IIT Indore 5
First generation sequencing

6. 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

7. Sanger method
5/29/2014 IIT Indore 7
http://bit.ly/1g6Cudq
http://bit.ly/1lcQO4J

8. Maxam-Gilbert method
5/29/2014 IIT Indore 8

9. Maxam-Gilbert method
5/29/2014 IIT Indore 9
http://bit.ly/1noY0fu
http://bit.ly/1lGvJCA

10. First generation sequencing
• Very high quality sequences (99.999%)
• Very low throughput
5/29/2014 IIT Indore 10
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

11. Next generation sequencing
5/29/2014 IIT Indore 11

12. 5/29/2014 IIT Indore 12
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

13. 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
5/29/2014 IIT Indore 13
http://www.acgt.me/blog/2014/3/10/next-generation-
sequencing-must-diepart-2

14. 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

15. Illumina
5/29/2014 IIT Indore 15
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

16. Illumina
5/29/2014 IIT Indore 16
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??

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

18. Illumina:Moleculo
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http://bit.ly/1aEPOBn

19. Pacific Biosciences SMRT sequencing
Single Molecule Real
Time sequencing
5/29/2014 IIT Indore 19
http://bit.ly/1naxgTe

20. Pacific Biosciences SMRT sequencing
Error correction methods
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Hierarchical genome-assembly
process (HGAP)
PBJelly
Enlish et al., PLOS One. 2012
PBJelly

21. 5/29/2014 IIT Indore 21
Pacific Biosciences SMRT sequencing
Read Lengths
http://www.igs.umaryland.edu/labs/grc/
Mean Read Length: 8391 bp
Maximum Subread Length: 24585 bp

22. Oxford Nanopore
5/29/2014 IIT Indore 22
https://www.nanoporetech.com/
• No data yet
• Error model
http://erlichya.tumblr.com/post/66376172948/hands-on-
experience-with-oxford-nanopore-minion

23. Others
• Ion Torrent Proton/PGM
• Nabsys
• SOLiD
5/29/2014 IIT Indore 23

24. Comparison
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25. 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

26. http://omicsmaps.com/
Next Generation Genomics:
World Map of High-throughput Sequencers
IIT Indore5/29/2014 26

27. 5/29/2014 IIT Indore 27
http://bit.ly/18pfUId

28. 5/29/2014 IIT Indore 28
http://bit.ly/18pfUId

29. Real cost of Sequencing!!
Sboner, Genome Biology, 2011
IIT Indore5/29/2014 29

30. 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
FR Illumina
Illumina
Slide credit: Aureliano Bombarely

31. Implications of Choice of Library
5/29/2014 IIT Indore 31
Slide credit: Aureliano Bombarely
Consensus sequence
(Contig)
Reads
Scaffold
(or Supercontig)
Pair Read information
NNNNN
Pseudomolecule
(or ultracontig)
F
Genetic information (markers)
NNNNN NN

32. 5/29/2014 IIT Indore 32
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

33. Which technology to use??
• Microbial genomes
• Eukaryotic genomes
• Resequencing genomes
• RNAseq and other XXXseq methods
5/29/2014 IIT Indore 33
http://bit.ly/1ko9Kgh

34. Looking into the Crystal ball
• Desktop sequencing
• Diagnostics in the clinic
• Large scale environmental sequencing of
microbes
• But challenges remain..
5/29/2014 IIT Indore 34

35. • 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/#!for
um/compbio_discussion
5/29/2014 IIT Indore 35

36. 5/29/2014 IIT Indore 36
• Collaborate with student
organizations
• Organize workshops and
journal clubs
• Attend international
meetings

37. 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

38. Thank you!!
Questions??
5/29/2014 BTI Plant Bioinformatics Course 2014 38