This was presented on Mar 31, 2015 at Boyce Thompson Institute, Ithaca, NY at the 3rd BTI Bioinformatics Course http://btiplantbioinfocourse.wordpress.com/

1. Surya Saha
Sol Genomics Network (SGN)
Boyce Thompson Institute, Ithaca, NY
ss2489@cornell.edu // Twitter:@SahaSurya
BTI Plant Bioinformatics Course 2015
http://www.acgt.me/blog/2015/3/7/next-generation-sequencing-must-die

2. 1953
DNA
Structure
discovery
1977
2012
Sanger DNA sequencing by
chain-terminating inhibitors
1984
Epstein-Barr
virus
(170 Kb)
1987
Abi370
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
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Pinus
taeda
(24 Gb)
2014
Nanopore
MinION

3. First generation sequencing
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Sanger. Annu Rev Biochem. 1988;57:1-28.
Thanks to Nick Loman for the mention

4. Maxam-Gilbert method
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5. Maxam-Gilbert method
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http://en.wikipedia.org/wiki/File:Maxam-
Gilbert_sequencing_en.svg
https://www.nationaldiagnostics.com/electrophoresis
/article/maxam-gilbert-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
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http://en.wikipedia.org/wiki/File:Sanger-sequencing.svg
http://en.wikipedia.org/wiki/File:
Radioactive_Fluorescent_Seq.jpg

8. First generation sequencing
• Very high quality sequences (99.999% or Q50)
• 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 384 1.9-84 Kb $2400
http://www.hindawi.com/journals/bmri/2012/251364/tab1/

9. Next generation sequencing
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10. 3/31/2015 BTI Plant Bioinformatics Course 2015 10
https://twitter.com/kbradnam/status/443153578429923328
• Second generation
• Third generation
• Fourth generation
• Next-next-generation
• Next-next-next
generation
http://www.acgt.me/blog/2015/3/10/next-generation-sequencing-must-diepart-2

11. Use the specific technology used
to generate the data
– Illumina Hiseq/Miseq/NextSeq
– Pacific Biosciences RS1/RSII
– Ion Torrent Proton/PGM
– SOLiD
– Oxford Nanopore
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http://www.acgt.me/blog/2015/3/10/next-generation-sequencing-must-
diepart-2

12. 454 Pyrosequencing
One purified DNA
fragment, to one bead, to
one read.
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http://www.genengnews.com/
GS FLX
Titanium
https://mariamuir.com/wp-
content/uploads/2013/04/rip.gif

13. Illumina
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Output 0.3-15 Gb 20-120 GB 10-1500 GB 900-1800 GB
Number
of Reads/
Flow cell
25 Million 130-400 Million 300 million – 2.5 Billion 3 Billion
Read
Length
2x300 bp 2x150 bp 2x250 - 2x125 bp 2x150 bp
Cost $99K $250K $740K $10M (10 units)
Source: Illumina
2500
3000
4000
500

14. Illumina
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Output 0.3-15 Gb 20-120 GB 10-1500 GB 900-1800 GB
Number
of Reads/
Flow cell
25 Million 130-400 Million 300 million – 2.5 Billion 3 Billion
Read
Length
2x300 bp 2x150 bp 2x250 - 2x125 bp 2x150 bp
Cost $99K $250K $740K $10M (10 units)
Source: Illumina
2500
3000
4000
$1000 human
genome??
500

15. Illumina
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Mardis 2008. Annu. Rev. Genomics Hum. Genet. 2008. 9:387–402

16. Illumina
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Mardis 2008. Annu. Rev. Genomics Hum. Genet. 2008. 9:387–402

17. Illumina:TruSeqLongRead
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Voskoboynik eLife 2013;2:e00569

18. Pacific Biosciences SMRT sequencing
Single Molecule Real
Time sequencing
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http://smrt.med.cornell.edu/images/pacbio_library_prep-1.gif

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

20. Pacific Biosciences SMRT sequencing
Error correction methods
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PBcRPipeline

21. 3/31/2015 BTI Plant Bioinformatics Course 2015 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. 3/31/2015 Centre for Agricultural Bioinformatics, Pusa 22
Pacific Biosciences SMRT sequencing
Read Lengths

23. Oxford Nanopore
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https://www.nanoporetech.com/
http://erlichya.tumblr.com/post/66376172948/hands-on-
experience-with-oxford-nanopore-minion
http://halegrafx.com/vector-art/free-vector-despicable-me-minions/

24. 3/31/2015 BTI Plant Bioinformatics Course 2015 24

25. Sequencing Trends
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https://www.google.com/trends/

26. 3/31/2015 BTI Plant Bioinformatics Course 2015 26
0
5000
10000
15000
20000
25000
30000
2008 2009 2010 2011 2012 2013 2014
Number of Publications
Illumina Pacific Biosciences Roche 454 Ion Torrent
-2000
-1000
0
1000
2000
3000
4000
5000
6000
2009 2010 2011 2012 2013 2014
Increase in Number of Publications
Illumina Pacific Biosciences Roche 454 Ion Torrent
0.00%
20.00%
40.00%
60.00%
80.00%
100.00%
120.00%
2009 2010 2011 2012 2013 2014
% Increase in Number of Publications
Pacific Biosciences Roche 454 Ion Torrent

27. Hi-C Crosslinking
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28. Others
• Ion Torrent Proton/PGM
• SOLiD
• Helicos
• Supporting technologies
– BioNano
– Nabsys
– OpGen
– 10X Genomics
– Fluidigm
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29. Comparison
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30. Next generation sequencing
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Run Time Read Length Quality
Total
nucleotides
sequenced
Cost /MB
454
Pyrosequencing
24h 700 bp Q20-Q30 1 GB $10
Illumina Miseq 27h 2x300bp > Q30 15 GB $0.15
Illumina Hiseq
2500
1 - 10days 2x250bp >Q30 3000 GB $0.05
Ion torrent 2h 400bp >Q20 50MB-1GB $1
Pacific
Biosciences
30m - 4h 10kb - >40kb
>Q50 consensus
>Q10 single
500 - 1000MB
/SMRT cell
$0.13 - $0.60
http://www.hindawi.com/journals/bmri/2012/251364/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431227

31. http://omicsmaps.com/
Next Generation Genomics:
World Map of High-throughput Sequencers
BTI Plant Bioinformatics Course 20153/31/2015 31

32. 3/31/2015 BTI Plant Bioinformatics Course 2015 32
https://flxlexblog.wordpress.com/2014/06/11/developments-in-next-generation-sequencing-june-2014-edition/

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https://flxlexblog.wordpress.com/2014/06/11/developments-in-next-generation-sequencing-june-2014-edition/

34. Real cost of Sequencing!!
Sboner, Genome Biology, 2011
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35. 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
BTI Plant Bioinformatics Course 2015

36. Implications of Choice of Library
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Slide credit: Aureliano Bombarely
Consensus sequence
(Contig)
Reads
Scaffold
(or Supercontig)
Pair Read information
NNNNN
Pseudomolecule
(or ultracontig)
F
Genetic information (markers) or Optical maps
NNNNN NN
BTI Plant Bioinformatics Course 2015

37. Multiplexing Libraries
Use of different tags (4-6 nucleotides) to identify
different samples in the same lane/sector.
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Slide credit: Aureliano Bombarely
AGTCGT
TGAGCA
AGTCGT
AGTCGT
AGTCGT
AGTCGT
TGAGCA
TGAGCA
TGAGCA
TGAGCA
AGTCGT
AGTCGT
AGTCGT
AGTCGT
TGAGCA
TGAGCA
TGAGCA
TGAGCA
Sequencing
BTI Plant Bioinformatics Course 2015

38. Fasta files:
It is a text-based format for representing either nucleotide sequences or peptide
sequences, in which nucleotides or amino acids are represented using single-letter codes.
-Wikipedia
File Formats
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Slide credit: Aureliano Bombarely
BTI Plant Bioinformatics Course 2015

39. Fastq files:
FASTQ format is a text-based format for storing both a biological sequence (usually
nucleotide sequence) and its corresponding quality scores.
-Wikipedia
• Single line ID with at symbol (“@”) in the first column.
• Sequences can be in multiple lines after the ID line
• Single line with plus symbol (“+”) in the first column to represent the quality line.
• Quality ID line may contain ID
• Quality values are in multiple lines after the + line but length is identical to sequence
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Slide credit: Aureliano Bombarely
File Formats
BTI Plant Bioinformatics Course 2015

40. 3/31/2015 40
Quality control: Encoding
Fastq files:
!"#$%&'()*+,-./0123456789 Offset by 33 (Phred+33)
KLMNOPQRSTUVWXYZ[]^_`abcdefgh Offset by 64 (Phred+64)
BTI Plant Bioinformatics Course 2015

41. Quality control: Encoding
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!"#$%&'()*+,-./0123456789 Offset by 33 (Phred+33)
KLMNOPQRSTUVWXYZ[]^_`abcdefgh Offset by 64 (Phred+64)
BTI Plant Bioinformatics Course 2015

42. 3/31/2015 42
Quality control: Encoding
http://en.wikipedia.org/wiki/Phred_quality_score
Phred score of a base is:
Qphred = -10 log10 (e)
where e is the estimated probability of a base
being wrong
BTI Plant Bioinformatics Course 2015

43. Pre-processing: Tools
Trimming
• FastQC
• FASTX toolkit
• Trimmomatic
• Scythe
Joining paired-end reads
• fastq-join
• FLASH
• PANDAseq
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44. Thank you!!
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