next-generation-sequencing-must-die surya saha · next generation sequencing 3/31/2015 bti plant...
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Surya Saha
Sol Genomics Network (SGN)
Boyce Thompson Institute, Ithaca, [email protected] // Twitter:@SahaSurya
BTI Plant Bioinformatics Course 2015
http://www.acgt.me/blog/2015/3/7/next-generation-sequencing-must-die
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DNA Structure discovery
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77
20
12
Sanger DNA sequencing by chain-terminating inhibitors
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84
Epstein-Barr virus
(170 Kb)
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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
Haemophilusinfluenzae(1.83 Mb)
20
13
Slide credit: Aureliano Bombarely
Sequencing over the Ages
Illumina
IlluminaHiseq X
454
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Pinustaeda
(24 Gb)
20
14
NanoporeMinION
First generation sequencing
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Sanger. Annu Rev Biochem. 1988;57:1-28.
Thanks to Nick Loman for the mention
Maxam-Gilbert method
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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
Sanger method
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Frederick Sanger13 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://en.wikipedia.org/wiki/File:Sanger-sequencing.svg
http://en.wikipedia.org/wiki/File:Radioactive_Fluorescent_Seq.jpg
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/
Next generation sequencing
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https://twitter.com/kbradnam/status/443153578429923328
• Second generation• Third generation• Fourth generation• Next-next-generation• Next-next-next
generationhttp://www.acgt.me/blog/2015/3/10/next-generation-sequencing-must-diepart-2
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
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
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
250030004000
500
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
250030004000
$1000 human genome??
500
Illu
min
a
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Mardis 2008. Annu. Rev. Genomics Hum. Genet. 2008. 9:387–402
Illu
min
a
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Mardis 2008. Annu. Rev. Genomics Hum. Genet. 2008. 9:387–402
Illu
min
a: T
ruSe
qLo
ng
Rea
d
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Voskoboynik eLife 2013;2:e00569
Pacific Biosciences SMRT sequencing
Single Molecule Real Time sequencing
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http://smrt.med.cornell.edu/images/pacbio_library_prep-1.gif
Pacific Biosciences SMRT sequencingError correction methods
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Hierarchical genome-assembly process (HGAP)
English et al., PLOS One. 2012
PBJelly
Pacific Biosciences SMRT sequencingError correction methods
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PB
cRP
ipel
ine
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Pacific Biosciences SMRT sequencingRead Lengths
http://www.igs.umaryland.edu/labs/grc/
Mean Read Length: 8391 bpMaximum Subread Length: 24585 bp
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Pacific Biosciences SMRT sequencingRead Lengths
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/
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Sequencing Trends
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https://www.google.com/trends/
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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
Hi-C Crosslinking
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Others
• Ion Torrent Proton/PGM
• SOLiD
• Helicos
• Supporting technologies– BioNano
– Nabsys
– OpGen
– 10X Genomics
– Fluidigm
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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
Pyrosequencing24h 700 bp Q20-Q30 1 GB $10
Illumina Miseq 27h 2x300bp > Q30 15 GB $0.15
Illumina Hiseq
25001 - 10days 2x250bp >Q30 3000 GB $0.05
Ion torrent 2h 400bp >Q20 50MB-1GB $1
Pacific
Biosciences30m - 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
http://omicsmaps.com/
Next Generation Genomics: World Map of High-throughput Sequencers
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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/
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
FR Illumina
Illumina
Slide credit: Aureliano BombarelyBTI Plant Bioinformatics Course 2015
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) or Optical maps
NNNNN NN
BTI Plant Bioinformatics Course 2015
Multiplexing Libraries
Use of different tags (4-6 nucleotides) to identify different samples in the same lane/sector.
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AGTCGT
TGAGCA
AGTCGTAGTCGT
AGTCGTAGTCGT
TGAGCATGAGCA
TGAGCATGAGCA
AGTCGT
AGTCGT
AGTCGT
AGTCGT
TGAGCATGAGCA
TGAGCA
TGAGCA
Sequencing
BTI Plant Bioinformatics Course 2015
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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BTI Plant Bioinformatics Course 2015
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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File Formats
BTI Plant Bioinformatics Course 2015
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Quality control: EncodingFastq files:
!"#$%&'()*+,-./0123456789 Offset by 33 (Phred+33)
KLMNOPQRSTUVWXYZ[\]^_`abcdefgh Offset by 64 (Phred+64)
BTI Plant Bioinformatics Course 2015
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
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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
Pre-processing: Tools
Trimming
• FastQC
• FASTX toolkit
• Trimmomatic
• Scythe
Joining paired-end reads
• fastq-join
• FLASH
• PANDAseq
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Thank you!!
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