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This was presented on Mar 11, 2014 at Boyce Thompson Institute, Ithaca, NY at the 3rd BTI Bioinformatics Course http://btiplantbioinfocourse.wordpress.com/
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Surya Saha
Sol Genomics Network (SGN)
Boyce Thompson Institute, Ithaca, NY [email protected] // Twitter:@SahaSurya
BTI Plant Bioinformatics Course 2014
http://www.acgt.me/blog/2014/3/7/next-generation-sequencing-must-die
19
53
DNA Structure discovery
19
77
20
12
Sanger DNA sequencing by chain-terminating inhibitors
19
84
Epstein-Barr virus
(170 Kb)
19
87
Abi370
Sequencer
19
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)
First generation sequencing
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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 RS1/RSII
– Ion Torrent Proton/PGM
– SOLiD
3/12/2014 BTI Plant Bioinformatics Course 2014 11
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
3/12/2014 BTI Plant Bioinformatics Course 2014 14
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
3/12/2014 BTI Plant Bioinformatics Course 2014 15 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
Others
• Ion Torrent Proton/PGM
• Oxford Nanopore
• 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
Summary
• Microbial genomes
• Eukaryotic genomes
• Resequencing genomes
• RNAseq and other XXXseq methods
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http://bit.ly/1ko9Kgh
http://omicsmaps.com/
Next Generation Genomics: World Map of High-throughput Sequencers
BTI Plant Bioinformatics Course 2014 3/12/2014 24
Real cost of Sequencing!!
Sboner, Genome Biology, 2011
BTI Plant Bioinformatics Course 2014 3/12/2014 27
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
3/12/2014 BTI Plant Bioinformatics Course 2014 29 Slide credit: Aureliano Bombarely
Consensus sequence
(Contig)
Reads
Scaffold
(or Supercontig)
Pair Read information
NNNNN
Pseudomolecule
(or ultracontig)
F
Genetic information (markers)
NNNNN NN
Multiplexing Libraries
Use of different tags (4-6 nucleotides) to identify different samples in the same lane/sector.
3/12/2014 BTI Plant Bioinformatics Course 2014 30 Slide credit: Aureliano Bombarely
AGTCGT
TGAGCA
AGTCGT AGTCGT
AGTCGT AGTCGT
TGAGCA TGAGCA
TGAGCA TGAGCA
AGTCGT
AGTCGT
AGTCGT
AGTCGT
TGAGCA TGAGCA
TGAGCA
TGAGCA
Sequencing
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
3/12/2014 BTI Plant Bioinformatics Course 2014 31 Slide credit: Aureliano Bombarely
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 should be identical to sequence
3/12/2014 BTI Plant Bioinformatics Course 2014 32 Slide credit: Aureliano Bombarely
File Formats
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Quality control: Encoding Fastq files:
!"#$%&'()*+,-./0123456789 Offset by 33 (Phred+33)
KLMNOPQRSTUVWXYZ[\]^_`abcdefgh Offset by 64 (Phred+64)
Quality control: Encoding
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!"#$%&'()*+,-./0123456789 Offset by 33 (Phred+33)
KLMNOPQRSTUVWXYZ[\]^_`abcdefgh Offset by 64 (Phred+64)
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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 wrong
Quality control: Error correction
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Thank you!!
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