Sequencing 2016

Surya Saha
Sol Genomics Network (SGN)
Boyce Thompson Institute, Ithaca, NY
suryasaha@cornell.edu // Twitter:@SahaSurya
BTI Plant Bioinformatics Course 2016
http://www.acgt.me/blog/2015/3/7/next-generation-sequencing-must-die

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 concept: Aureliano Bombarely
Sequencing over the Ages
Illumina
Illumina
Hiseq X
454
3/29/2016 BTI Plant Bioinformatics Course 2016 2
Pinus
taeda
(24 Gb)
2014
Nanopore
MinION
2015
10X
Genomics

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

Maxam-Gilbert method

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

Sanger method
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
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 very low throughput
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

Use the specific technology used
to generate the data
– Illumina Hiseq/Miseq/NextSeq
– Pacific Biosciences RS I/RS II
– Ion Torrent Proton/PGM
– SOLiD
– Oxford Nanopore
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.
http://www.genengnews.com/
GS FLX
Titanium
https://mariamuir.com/wp-
content/uploads/2013/04/rip.gif

Illumina
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

Illumina
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

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

Illumina:TruSeqLongRead
Voskoboynik eLife 2013;2:e00569

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

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

Error correction methods
PBcRPipeline

3/29/2016 Centre for Agricultural Bioinformatics, Pusa 20
Read Lengths

Oxford Nanopore
3/29/2016 Centre for Agricultural Bioinformatics, Pusa 21
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/

Next generation sequencing
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
Note: Some figures might be out of date

Long range scaffolding

Hi-C Crosslinking

http://mms.businesswire.com/media/20150225005296/en
/454639/5/GemCodePlatform.jpg
• Long read information from short reads
using 14bp bar codes
• Very low input DNA (ng) and 20 minute
library preparation time
• 1ng of DNA is split across 100,000 Gel
Coated Beads (GEMs)
• Chromium instrument for single-cell
RNAseq
GemCode

http://mms.businesswire.com/media/20150225005296/en
/454639/5/GemCodePlatform.jpg
GemCode
http://www.nature.com/nbt/journal/v34/n3/full/nbt.3432.html

http://www.bionanogenomics.com/technology/why-genome-mapping/

Human MHC map
• Sample prep requires very high molecular weight DNA
• Nicks at 10 sites / 100kb
• Individual molecules are assembles into optical maps
• Optical maps and sequences are merged in a hybrid assembly
http://www.bionanogenomics.com/technology/why-genome-mapping/

Many Others..
• Ion Torrent Proton/PGM
• Supporting technologies
– Nabsys
– OpGen
– Fluidigm
http://nextgenseek.com/2012/11/did-you-know-there-are-
at-least-14-next-gen-sequence-technology-companies/

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

0
5000
10000
15000
20000
25000
30000
35000
2008 2009 2010 2011 2012 2013 2014 2015
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 2015
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 2015
% Increase in Number of Publications
Pacific Biosciences Roche 454 Ion Torrent

Real cost of Sequencing!!
Sboner, Genome Biology, 2011
3/29/2016 33BTI Plant Bioinformatics Course 2016

https://genomebiology.biomedcentral.com/articles/10.1186/gb-2011-12-8-125

So What Sequencer Do I Use??
Microbial genome
• Draft genome
– Illumina Miseq (100-130X)
– Illumina Hiseq (<200X)
• Complete genome
– Pacific Biosciences (80-100X)
• Amplicons (16S, ITS)
– Illumina Miseq
Eukaryotic genome
• Denovo assembly
– Pacific Biosciences (70-80X)
– Illumina Hiseq (100X+)
– 10X Genomics
– Bionano
• Genotyping (GBS)
– Illumina Hiseq
• BACs
– Pacific Biosciences
$$$$ ????

The diploid
reference
genome

Cornell Sequencing Core
• Illumina Hiseq 2500 (Rapid run and High output)
• Illumina Miseq
• Illumina Nextseq 500
• 10X Genomics GemCode
http://www.biotech.cornell.edu/brc/g
enomics/services/price-list#overlay-
context=brc/genomics-facility/next-
generation-sequencing
$
$
$

Library Types
Single end
Pair end (PE, 150-300 bp, Fwd:/1, Rev:/2)
Mate pair (MP, 2Kb to 20 Kb)
3/29/2016 38
F
F R
F R 454/Roche
FR Illumina
Illumina
Slide credit: Aureliano Bombarely

Implications of Choice of Library
3/29/2016 39
Consensus sequence
(Contig)
Reads
Scaffold
(or Supercontig)
Pair Read information
NNNNN
Pseudomolecule
(or ultracontig)
F
Genetic information (markers) or Optical maps
NNNNN NN

Multiplexing Libraries
Use of different tags (4-6 nucleotides) to identify
different samples in the same lane/sector.
3/29/2016 40
AGTCGT
TGAGCA
AGTCGT
AGTCGT
AGTCGT
AGTCGT
TGAGCA
TGAGCA
TGAGCA
TGAGCA
AGTCGT
AGTCGT
AGTCGT
AGTCGT
TGAGCA
TGAGCA
TGAGCA
TGAGCA
Sequencing

Data!!

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
3/29/2016 42

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
3/29/2016 43
File Formats

3/29/2016 44
Quality control: Encoding
Fastq files:
!"#$%&'()*+,-./0123456789 Offset by 33 (Phred+33)
KLMNOPQRSTUVWXYZ[]^_`abcdefgh Offset by 64 (Phred+64)

3/29/2016 45
!"#$%&'()*+,-./0123456789 Offset by 33 (Phred+33)
KLMNOPQRSTUVWXYZ[]^_`abcdefgh Offset by 64 (Phred+64)

3/29/2016 46
http://en.wikipedia.org/wiki/Phred_quality_score
Phred score of a base is:
Qphred = -10 log10 (e)
where e is the estimated error probability of a base

Pre-processing: Tools
Trimming
• FastQC
• FASTX toolkit
• Trimmomatic
• Scythe
Joining paired-end reads
• fastq-join
• FLASH
• PANDAseq
3/29/2016 47BTI Plant Bioinformatics Course 2016

Thank you!!

Sequencing 2016

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Sequencing 2016