IMGS 2011 Bioinformatics Workshop
Deanna Church, NCBI
Carol Bult, The Jackson Laboratory
Intro
Sequencing Technology: life in the fast laneAlignments: things to considerFile formats: everything you always wanted to know but were afraid to askTools: Pick the right one for the job at hand
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Gig
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esCost per Kb
Lucinda Fulton, The Genome Center at Washington University
Cost Throughput
Sequencing Technologies
http://www.geospiza.com/finchtalk/uploaded_images/plates-and-slides-718301.png
Sequence “Space”• Roche 454 – Flow space
– Measure pyrophosphate released by a nucleotide when it is added to a growing DNA chain
– Flow space describes sequence in terms of these base incorporations– http://www.youtube.com/watch?v=bFNjxKHP8Jc
• AB SOLiD – Color space– Sequencing by DNA ligation via synthetic DNA molecules that contain two nested known
bases with a flouorescent dye– Each base sequenced twice– http://www.youtube.com/watch?v=nlvyF8bFDwM&feature=related
• Illumina/Solexa – Base space– Single base extentions of fluorescent-labeled nucleotides with protected 3 ‘ OH groups– Sequencing via cycles of base addition/detection followed deprotection of the 3’ OH– http://www.youtube.com/watch?v=77r5p8IBwJk&feature=related
• GenomeTV – Next Generation Sequencing (lecture)– http://www.youtube.com/watch?v=g0vGrNjpyA8&feature=related
http://finchtalk.geospiza.com/2008/03/color-space-flow-space-sequence-space_23.html
Global and local alignments
Optimal global alignment
Needleman-Wunsch
Sequences align essentially from end to end
Optimal local alignment
Smith-Waterman
Sequences align only in small, isolated regions
References
Needleman and Wunsch (1970). J. Mol. Biol. 48, 443-453.
Smith and Waterman (1981). Nucleic Acids Res 13, 645-656.
Hashing methods
MVRRLPERTSTPACE
MVRVRRRRLRLPLPEPERERTRTSTSTSTPTPAPACACE
Query sequence
Word size = 3(configurable)
References
Wilbur & Lipman (1983), PNAS 80, 726-30
Lipman & Pearson (1985), Science 227, 1435-1441
Pearson & Lipman (1988), PNAS 85, 2444-2448
http://www.slideshare.net/thomaskeane/eccb-2010-nextgen-sequencing-tutorial
http://www.slideshare.net/thomaskeane/eccb-2010-nextgen-sequencing-tutorial
Sensitivity vs. Specificity
Sensitivity = actual number of true positives (tp) identifiedSpecificity = number of true negatives (tn) identified
Actu
al
Predicted
TP FN
FP TN
positives
negatives
positives negatives
Sensitivity= TP/(TP+FN)Specificity=TN/(TN+FP)
Richa Agarwala
MHC Alternate locus
Alignment to chr6
ToolsAlignments
BLAST: not for NGSBWABowtieMaq…
TranscriptomicsTophatCufflinks…
Variant callingssahaSNPMosaic…
Counting (Chip-Seq, etc)FindPeaksPeakSeq
Genome Workbenchhttp://www.ncbi.nlm.nih.gov/projects/gbench/
“Standard” File formats
Sequence containersFASTAFASTQBAM/SAM
AlignmentsBAM/SAMMAF
AnnotationBEDGFF/GTF/GFF3WIG
VariationVCFGVF
FASTQ: Data Format• FASTQ
– Text based– Encodes sequence calls and quality scores with ASCII characters– Stores minimal information about the sequence read– 4 lines per sequence
• Line 1: begins with @; followed by sequence identifier and optional description
• Line 2: the sequence• Line 3: begins with the “+” and is followed by sequence identifiers and
description (both are optional)• Line 4: encoding of quality scores for the sequence in line 2
• References/Documentation– http://maq.sourceforge.net/fastq.shtml– Cock et al. (2009). Nuc Acids Res 38:1767-1771.
FASTQ Example
FASTQ example from: Cock et al. (2009). Nuc Acids Res 38:1767-1771.
For analysis, it may be necessary to convert to the Sanger form of FASTQ…For example,
Illumina stores quality scores ranging from 0-62;Sanger quality scores range from 0-93.
Solexa quality scores have to be converted to PHRED quality scores.
SAM (Sequence Alignment/Map)
• It may not be necessary to align reads from scratch…you can instead use existing alignments in SAM format– SAM is the output of aligners that map reads to a
reference genome– Tab delimited w/ header section and alignment
section• Header sections begin with @ (are optional)• Alignment section has 11 mandatory fields
– BAM is the binary format of SAM
http://samtools.sourceforge.net/
http://samtools.sourceforge.net/SAM1.pdf
Mandatory Alignment Fields
http://samtools.sourceforge.net/SAM1.pdf
Alignment Examples
Alignments in SAM format
chr1 86114265 86116346 nsv433165chr2 1841774 1846089 nsv433166chr16 2950446 2955264 nsv433167chr17 14350387 14351933 nsv433168chr17 32831694 32832761 nsv433169chr17 32831694 32832761 nsv433170chr18 61880550 61881930 nsv433171
chr1 16759829 16778548 chr1:21667704 270866 -chr1 16763194 16784844 chr1:146691804 407277 +chr1 16763194 16784844 chr1:144004664 408925 -chr1 16763194 16779513 chr1:142857141 291416 -chr1 16763194 16779513 chr1:143522082 293473 -chr1 16763194 16778548 chr1:146844175 284555 -chr1 16763194 16778548 chr1:147006260 284948 -chr1 16763411 16784844 chr1:144747517 405362 +
Valid BED files
Mouse chrX: 35,000,000-36,000000
Mouse chrX: 35,000,000-36,000000
X
MGSCv3 Build 36
NC_000086.6
hg19GRCh37
mm8MGSCv37
NCBIM37
danRer5Zv7
Assemblies with the same name aren’t always the same
chr21:8,913,216-9,246,964
Assemblies with the same name aren’t always the same
Zv7 chr21:8,913,216-9,246,964 X Mouse Build 36 chrX
hg19GRCh37
GCA_000001405.1
Tutorial Web Sitehttp://www.ncbi.nlm.nih.gov/staff/church/GenomeAnalysis/index.shtml
This site will be accessible after the meeting. Check back for updates and new tutorials.
RNA Seq Workflow• Convert data to FASTQ• Upload files to Galaxy• Quality Control
– Throw out low quality sequence reads, etc.• Map reads to a reference genome
– Many algorithms available– Trade off between speed and sensitivity
• Data summarization– Associating alignments with genome annotations– Counts
• Data Visualization• Statistical Analysis
Typical RNA_Seq Project Work Flow
Sequencing Sequencing
Tissue Sample Tissue Sample
Cufflinks Cufflinks
TopHat TopHat
FASTQ file FASTQ file
QC QC
Gene/Transcript/Exon Expression
Gene/Transcript/Exon Expression
VisualizationVisualization
Total RNA Total RNA mRNA mRNA cDNA cDNA
Statistical Analysis
Statistical Analysis
JAX Computational Sciences Service
TopHat
Trapnell et al. (2009). Bioinformatics 25:1105-1111.
http://tophat.cbcb.umd.edu/
Figure from: Trapnell et al. (2010). Nature Biotechnology 28:511-515.
TopHat is a good tool for aligning RNA Seq data compared to other aligners (Maq, BWA) because it takes splicing into account during the alignment process.
Trapnell C et al. Bioinformatics 2009;25:1105-1111
TopHat is built on the Bowtie alignment algorithm.
Cufflinks
Trapnell et al. (2010). Nature Biotechnology 28:511-515.
http://cufflinks.cbcb.umd.edu/
• Assembles transcripts,• Estimates their abundances, and •Tests for differential expression and regulation in RNA-Seq samples
Galaxyhttp://main.g2.bx.psu.edu/
See Tutorial 1
Build and share data and analysis workflowsNo programming experience requiredStrong and growing development and user community
Short Read Archivehttp://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?
Short Read Archive Handbookhttp://www.ncbi.nlm.nih.gov/books/NBK47528/
http://www.asperasoft.com/en/products/client_software_2/aspera_connect_8
High performance file transfer for getting data from the Short Read Archive
Aspera Connect
http://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=softwareSRA Toolkit
Galaxy on the Cloud• Create an Amazon Web Services AWS account
– Sign up for Amazon Elastic Compute Cloude (EC2) and– Amazon Simple Storage Service (S3 service)
• Use the AWS Management Console to start a master EC2 instance
• Use the Galaxy Cloud web interface to manage the cluster• Step by step instructions are here:
– https://bitbucket.org/galaxy/galaxy-central/wiki/cloud
• Screencast to demonstrate the sign up process is here:– https://bitbucket.org/galaxy/galaxy-central/wiki/cloud
Afgan E., Baker D., Coraor N., Chapman B., Nekrutenko A., Taylor J. (2010) BMC Bioinformatics. 11:2010.
Why Go to the Cloud?• Files and Compute needs are much greater for next gen
sequence data • Amazon cloud provides a scalable, cost-effective solution
Afgan E., Baker D., Coraor N., Chapman B., Nekrutenko A., Taylor J. (2010) BMC Bioinformatics. 11:2010.
Some Tips
• You’ll need a credit card to activate the service • You’ll need to be near a phone so that you can
verify your identity during the sign up process• There is a time lag between signing up for
AWS and getting access
Tools HistoryDialog/Parameter Selection
Let’s Get Started!