surya saha - wordpress.com · 2018-04-03 · surya saha sol genomics network (sgn) boyce thompson...

Surya SahaSol Genomics Network (SGN)

Boyce Thompson Institute, Ithaca, [email protected] // Twitter:@SahaSurya

BTI Plant Bioinformatics Course 2018

http://www.acgt.me/blog/2015/3/7/next-generation-sequencing-must-die

mailto:[email protected]

https://twitter.com/SahaSurya

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

Haemophilusinfluenzae(1.83 Mb)

20

13

Slide concept: Aureliano Bombarely

Sequencing over the Ages

Illumina

IlluminaHiseq X

454

4/2/2018 BTI Plant Bioinformatics Course 2018 2

Pinustaeda

(24 Gb)

20

14

NanoporeMinION

20

15

10XGenomics

First generation sequencing


Sanger. Annu Rev Biochem. 1988;57:1-28.

Thanks to Nick Loman for the mention

Sanger method


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

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

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/

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/Novaseq

– Pacific Biosciences RS I/RS II/Sequel

– Ion Torrent Proton/PGM

– Oxford Nanopore


http://www.acgt.me/blog/2015/3/10/next-generation-sequencing-must-diepart-2

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.


http://www.genengnews.com/

GS FLX Titanium

https://mariamuir.com/wp-content/uploads/2013/04/rip.gif

http://www.genengnews.com/

https://mariamuir.com/wp-content/uploads/2013/04/rip.gif

Illumina


Output 15 Gb 120 GB 1500 GB 1800 GB

Max Number of Reads/ Run

25 Million 400 Million 5 Billion 6 Billion

Max Read Length

2x300 bp 2x150 bp 2x125- 2x250 bp (RR mode) 2x150 bp

Cost $99K $250K $740K $10M (10 units)

Source: Illumina

250030004000

500550

Illu

min

a


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

Pacific Biosciences SMRT sequencing

Single Molecule Real Time sequencing


http://smrt.med.cornell.edu/images/pacbio_library_prep-1.gif

RS II

Sequel

http://smrt.med.cornell.edu/images/pacbio_library_prep-1.gif

Pacific Biosciences SMRT sequencingError correction methods


PB

cRP

ipel

ine


Pacific Biosciences SMRT sequencingRead Lengths

Oxford Nanopore


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/

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/


http://lab.loman.net/2017/03/09/ultrareads-for-nanopore/

E. coli K-12 MG1655 on a standard FLO-MIN106 (R9.4) flowcell

http://lab.loman.net/2017/03/09/ultrareads-for-nanopore/

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 ( as low as 0.625 ng) • Short 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


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

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

Many Others..

• Ion Torrent Proton/PGM

• Dovetail

• Supporting technologies

– Nabsys

– OpGen

– Fluidigm


http://nextgenseek.com/2012/11/did-you-know-there-are-at-least-14-next-gen-sequence-technology-companies/

http://nextgenseek.com/2012/11/did-you-know-there-are-at-least-14-next-gen-sequence-technology-companies/

Real cost of Sequencing!!

Sboner, Genome Biology, 2011

4/2/2018 25BTI Plant Bioinformatics Course 2018

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

– Hi-C

• Genotyping (GBS)– Illumina Hiseq

• BACs– Pacific Biosciences


$$$$ ????

Genome Assembly


http://biobeans.blogspot.com/2012/11/bioinformatics-genome-assembly.html


Slide credit: Torsten Seemann

Whole Genome Shotgun Sequencing

4/3/2018 29Slide credit: cbcb.umd.edu


Genome Sequencing Strategies

4/3/2018 Centre for Agricultural Bioinformatics, Pusa 30

International Human Genome Sequencing Consortium 2001

Overlap Layout Consensus

http://contig.wordpress.com/

cbcb.umd.edu

Lon

g re

ad s

eq

uen

cin

g

Overlap-Layout-Consensus


Slide source: Commins 2009


De

Bru

ijn G

rap

h

Ingredients for a Good Assembly

4/3/2018 33

Slide credit: Mike Schatz



The diploid reference genome

CHROMOSOMES

SCAFFOLDSCONTIGS


Gene to Genome – The BIG picture

CONTIG GAPSSCAFFOLD GAPS

GENES

MAP (chr1)Ovate (chr1)TM (chr 9)L2 (chr 10)


State of the SL2.50 Build

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0 1 2 3 4 5 6 7 8 9 10 11 12

Sequence Scaffold gap length Component gap length

Length 823Mb

Sequence 737Mb

Contig gaps 43Mb (5.30%)

Scaffold gaps 42Mb (5.17%)

Total gaps 86Mb (10.47%)

Reference assembly but plenty of gaps!!


Summary

Any genome assembly:

• Is a hypothesis that needs to be refined

• Is a work in progress

• Can sometimes be misguiding

So is genome annotation…..


Gene structure improvement example

ITAG3.2

ITAG2.40

ITAG3.2

ITAG2.40Fusion of split genes

UTR extension

RNAseq

XY plot

RNAseq

XY plot

Required for 3’ RNAseq


Quality check - Annotation Edit Distance (AED)

Based on RNAseq data support

AED= 0 complete support

AED =1 lack of support

Annotation Edit Distance

AED provides a means to

evaluate quality of annotations

given RNAseq and ortholog

evidence

Cu

mu

lati

ve f

ract

ion

of

tran

scri

pts


Solanaceae Apollo annotation editor

Genomes available in Apollo • Request access to Apollo by

contacting SGN• More organisms will be added

as they become available.

For creating account: https://solgenomics.net/contact/form

Apollo: collaborative genome annotation editorhttps://github.com/gmod/apollohttp://genomearchitect.org

https://solgenomics.net/contact/form

https://github.com/gmod/apollo

http://genomearchitect.org/


Editing an existing gene model


Correction of predicted gene model


Information Editor• DBXRefs (InterPro, Pfam)• PubMed IDs• Gene Ontology IDs (GO)• Comments

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/genomics/services/price-list#overlay-context=brc/genomics-facility/next-generation-sequencing

$

$

$

http://www.biotech.cornell.edu/brc/genomics/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)

4/2/2018 45

F

F R

F R 454/Roche

FR Illumina

Illumina

Slide credit: Aureliano BombarelyBTI Plant Bioinformatics Course 2018

Implications of Choice of Library

4/2/2018 46Slide 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


Multiplexing Libraries

Use of different tags (4-6 nucleotides) to identify different samples in the same lane/sector.


AGTCGT

TGAGCA

AGTCGTAGTCGT

AGTCGTAGTCGT

TGAGCATGAGCA

TGAGCATGAGCA

AGTCGT

AGTCGT

AGTCGT

AGTCGT

TGAGCATGAGCA

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



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


File Formats


4/2/2018 51

Quality control: EncodingFastq files:

!"#$%&'()*+,-./0123456789 Offset by 33 (Phred+33)

KLMNOPQRSTUVWXYZ[\]^_`abcdefgh Offset by 64 (Phred+64)


Quality control: Encoding

4/2/2018 52

!"#$%&'()*+,-./0123456789 Offset by 33 (Phred+33)

KLMNOPQRSTUVWXYZ[\]^_`abcdefgh Offset by 64 (Phred+64)


4/2/2018 53

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 error probability of a base


http://en.wikipedia.org/wiki/Phred_quality_score

Pre-processing: Tools

Trimming

• FastQC

• FASTX toolkit

• Trimmomatic

• Scythe

Joining paired-end reads

• fastq-join

• FLASH

• PANDAseq


Thank you!!


surya saha - wordpress.com · 2018-04-03 · surya saha sol genomics network (sgn) boyce thompson...

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