Alignment - UC Davis Bioinformatics Core

Transcription

Alignment
UCD Genome Center Bioinformatics Core
Wednesday 17 June 2015
From reads to molecules
UC Davis Genome Center | Bioinformatics Core | J Fass
Alignment 2015-06-17
Why align?
Individual A
Individual B
ATGATAGCATCGTCGGGTGTCTGCTCAATAATAGTGCCGTATCATGCTGGTGTTATAATCGCCGCATGACATGATCAATGG
CAATAAAAGTGCCGTATCATGCTGGTGTTACAATCGCCGCA
CGTATCATGCTGGTGTTACAATCGCCGCATGACATGATCAATGG
TGTCTGCTCAATAAAAGTGCCGTATCATGCTGGTGTTACAATC
ATCGTCGGGTGTCTGCTCAATAAAAGTGCCGTATCATG--GGTGTTATAA
CTCAATAAGAGTGCCGTATCATG--GGTGTTATAATCGCCGCA
GTTATAATCGCCGCATGACATGATCAATGG
To measure variation.
Why align?
Why align?
Short Read Aligners (DNA, not RNA)
Fall '12 - Apr '13: ... now 150-180 Gbp / day!*
* http://www.illumina.com/systems/hiseq_2500_1500/performance_specifications.ilmn
Burrows-Wheeler Aligners
Burrows-Wheeler Transform used in bzip2 file
compression tool; FM-index (Ferragina & Manzini)
allow efficient finding of substring matches within
compressed text – algorithm is sub-linear with
respect to time and storage space required for a
certain set of input data (reference 'ome,
essentially).
Reduced memory footprint, faster execution.
BWA
BWA is fast, and can do gapped alignments. When run without
seeding, it will find all hits within a given edit distance. Long
read aligner is also fast, and can perform well for 454, Ion
Torrent, Sanger, and PacBio reads. BWA is actively developed
and has a strong user / developer community.
bio-bwa.sourceforge.net
Short reads – under 200 bp
Li H. and Durbin R. (2009) “Fast and accurate short read alignment with
Burrows-Wheeler Transform.” Bioinformatics, 25:1754-60. [PMID: 19451168]
Long reads – over 200 bp … chimeric alignments built-in
Li H. and Durbin R. (2010) “Fast and accurate long read alignment with
Burrows-Wheeler Transform.” Bioinformatics, 26:589-95. [PMID: 20080505]
… don't forget to join the mailing groups!
Bowtie
Bowtie (now Bowtie 2) is probably faster than BWA for some
types of alignment, but it may not find the best alignments (see
discussions on sensitivity, accuracy on SeqAnswers.com).
Bowtie is part of a suite of tools (Bowtie, Tophat, Cufflinks,
CummeRbund) that address RNAseq experiments.
http://bowtie-bio.sourceforge.net
Langmead B., Trapnell C., Pop M., and Salzberg S.L. (2009) “Ultrafast and
memory-efficient alignment of short DNA sequences to the human genome”
Genome Biology 10:R25 [PMID: 19261174]
… don't forget to join the mailing groups!
Alignment concepts / parameters
Paired-End reads
Mate-Paired reads
454 "Paired-End" reads
Single End Construct
File Format: SAM / BAM / CRAM
!
NEW
http://samtools.sourceforge.net/ - deprecated!
http://www.htslib.org/ - SAMtools 1.0 and up
Li H.*, Handsaker B.*, Wysoker A., Fennell T., Ruan J., Homer N.,
Marth G., Abecasis G., Durbin R. and 1000 Genome Project Data
Processing Subgroup (2009) The Sequence alignment/map (SAM)
format and SAMtools. Bioinformatics, 25, 2078-9. [PMID: 19505943]
●
●
●
●
SAM specification (currently v1, renumbered … 1 is after old v1.4)
samtools man page
example workflow(s)
mailing list!
File Format: SAM
File Format: SAM
SAM Format Specification v1.4-r985
7,8 - formerly MRNM,
MPOS (mate reference
name, mate position)
9 - formerly ISIZE ("insert" size)
File Format: SAM
google "Heng Li slides" - Challenges and Solutions in the Analysis of Next Generation Sequencing Data (2010)
File Format: BAM
BAMs are compressed SAMs (so, binary, not human-readable text …
don't look directly at them!). They can be indexed to allow rapid
extraction of information, so alignment viewers do not need to
uncompress the whole BAM file in order to look at information for a
particular read or coordinate range, somewhere in the file.
Indexing your BAM file, myCoolBamFile.bam, will create an index file,
myCoolBamFile.bam.bai, which is needed (in addition to the BAM file)
by viewers and other downstream tools. An occasional downstream tool
will require an index called myCoolBamFile.bai (notice that the “.bai”
replaces the “.bam”, instead of being appended after it).
File Format: CRAM
Available as of SAMtools 1.0, and is a binary format like BAM. Uses
data-specific compression tools (i.e. compressing letters is different
than compressing numbers), specifically reference-based compression
(e.g. for aligned reads, only mis-matching bases need to be stored).
Also can employ lossy compression of base qualities, which appears to
have a negligible effect on, say, variant calling (see Illumina white
paper).
Indexing your CRAM file, myCoolBamFile.cram, will create an index file,
myCoolBamFile.cram.crai, which is needed (in addition to the CRAM
file) by viewers and other downstream tools.
This is a very recent development, so it may be a while before tools
are CRAM-capable.
Alignment Viewers
●
●
●
IGV (Integrated Genomics Viewer)
○ www.broadinstitute.org/igv/
BAMview, tview (in SAMtools), IGB, GenomeView, SAMscope ...
UCSC Genome Browser, GBrowse
IGV
red box indicates region of
reference in view below
coverage track: read coverage depth
plot
read alignments: (various view styles - “squished” shown here) … read positions,
orientations, pairing, sequence that disagrees with reference highlighted, improper pairs
highlighted, etc.
annotation tracks (GTF, BED, etc.)
IGV
colored bases where they
disagree with reference
(substitution, indel, etc.)
improper pairs (mate
aligns far away, in wrong
orientation, or on another
chromosome)
reference sequence,
reading frames, etc.
Variant Calling - VCF format
One main application of read alignment. A.k.a. "resequencing", SNP /
indel discovery. VCF (variant call format) is now the standard format for
variant reporting.
http://vcftools.sourceforge.net/specs.html ... VCF poster
Variant Call Format
##fileformat=VCFv4.1
##fileDate=20130825
##source=freeBayes v9.9.2-9-gfbf46fc-dirty
##reference=../results/8/8.fa
##phasing=none
##commandline="../tools/freebayes/bin/freebayes -f ../results/8/8.fa --min-alternate-fraction 0.03 --minmapping-quality 20 --min-base-quality 20 --ploidy 1 --pooled-continuous --use-best-n-alleles 4 --usemapping-quality --min-alternate-fraction 0.04 --min-alternate-count 1 ../results/8/8.bam"
##INFO=<ID=RO,Number=1,Type=Integer,Description="Reference allele observation count, with partial
observations recorded fractionally">
##INFO=<ID=AO,Number=A,Type=Integer,Description="Alternate allele observations, with partial observations
recorded fractionally">
##INFO=<ID=TYPE,Number=A,Type=String,Description="The type of allele, either snp, mnp, ins, del, or
complex.">
##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
##FORMAT=<ID=GQ,Number=1,Type=Float,Description="Genotype Quality, the Phred-scaled marginal (or
unconditional) probability of the called genotype">
##FORMAT=<ID=GL,Number=G,Type=Float,Description="Genotype Likelihood, log10-scaled likelihoods of the data
given the called genotype for each possible genotype generated from the reference and alternate alleles
given the sample ploidy">
##FORMAT=<ID=DP,Number=1,Type=Integer,Description="Read Depth">
##FORMAT=<ID=RO,Number=1,Type=Integer,Description="Reference allele observation count">
##FORMAT=<ID=QR,Number=1,Type=Integer,Description="Sum of quality of the reference observations">
##FORMAT=<ID=AO,Number=A,Type=Integer,Description="Alternate allele observation count">
##FORMAT=<ID=QA,Number=A,Type=Integer,Description="Sum of quality of the alternate observations">
#CHROM POS
ID
REF
ALT
QUAL
FILTER INFO
FORMAT 8
8_PB1
26
.
TGTTACGCG
GCTTTTGC,TGTTTCTAC
27.2619 .
AO=1,2;RO=0;TYPE=complex,
complex
GT:DP:RO:QR:AO:QA:GL
2:3:0:0:1,2:31,70:-4.46,-1.65,0
8_PB1
38
.
TCA
ACG,TA,AGA
0.0495692
.
AO=1,1,1;RO=3;TYPE=complex,del,mnp
2:6:3:101:1,1,1:31,37,34:0,-4.556,-4.004,-4.28
8_PB1
42
.
G
A
3.94171e-14
.
AO=8;RO=128;TYPE=snp
GT:DP:RO:QR:AO:QA:
GL
Variant Call Format
##fileformat=VCFv4.1
##fileDate=20130825
##source=freeBayes v9.9.2-9-gfbf46fc-dirty
##reference=../results/8/8.fa
##phasing=none
##commandline="../tools/freebayes/bin/freebayes -f ../results/8/8.fa --min-alternate-fraction 0.03 --minmapping-quality 20 --min-base-quality 20 --ploidy 1 --pooled-continuous --use-best-n-alleles 4 --usemapping-quality --min-alternate-fraction 0.04 --min-alternate-count 1 ../results/8/8.bam"
##INFO=<ID=RO,Number=1,Type=Integer,Description="Reference allele observation count, with partial
##INFO=<ID=AO,Number=A,Type=Integer,Description="Alternate allele observations, with partial observations
recorded fractionally">
##INFO=<ID=TYPE,Number=A,Type=String,Description="The type of allele, either snp, mnp, ins, del, or
complex.">
##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
##FORMAT=<ID=GQ,Number=1,Type=Float,Description="Genotype Quality, the Phred-scaled marginal (or
##FORMAT=<ID=GL,Number=G,Type=Float,Description="Genotype Likelihood, log10-scaled likelihoods of the data
given the called genotype for each possible genotype generated from the reference and alternate alleles
given the sample ploidy">
##FORMAT=<ID=DP,Number=1,Type=Integer,Description="Read Depth">
##FORMAT=<ID=RO,Number=1,Type=Integer,Description="Reference allele observation count">
##FORMAT=<ID=QR,Number=1,Type=Integer,Description="Sum of quality of the reference observations">
##FORMAT=<ID=AO,Number=A,Type=Integer,Description="Alternate allele observation count">
##FORMAT=<ID=QA,Number=A,Type=Integer,Description="Sum of quality of the alternate observations">
#CHROM POS
ID
REF
ALT
QUAL
FILTER INFO
FORMAT 8
8_PB1
26
.
TGTTACGCG
GCTTTTGC,TGTTTCTAC
27.2619 .
AO=1,2;RO=0;TYPE=complex,
complex
2:3:0:0:1,2:31,70:-4.46,-1.65,0
8_PB1
38
.
TCA
ACG,TA,AGA
0.0495692
.
AO=1,1,1;RO=3;TYPE=complex,del,mnp
2:6:3:101:1,1,1:31,37,34:0,-4.556,-4.004,-4.28
8_PB1
42
.
G
A
3.94171e-14
.
GT:DP:RO:QR:AO:QA:
GL
Variant Call Format
#CHROM POS
ID
REF
8_PB2
407
.
A
170:21:788:149:5579:-5,0
CHROM
= 8_PB2
POS
= 407
ID
= .
REF
= A
ALT
= G
QUAL
= 3935.83
ALT
G
QUAL
FILTER
3935.83 .
INFO
FORMAT 8
1:
FILTER = .
INFO
= AO=149;RO=21;TYPE=snp
FORMAT = GT:DP:RO:QR:AO:QA:GL
8
= 1:170:21:788:149:5579:-5,0
Variant Call Format
Variant Call Format
Variant Call Format
Variant Call Format
#CHROM POS
ID
REF
8_PB2
407
.
A
170:21:788:149:5579:-5,0
CHROM
= 8_PB2
POS
= 407
ID
= .
REF
= A
ALT
= G
QUAL
= 3935.83
ALT
G
QUAL
FILTER
3935.83 .
INFO
FORMAT 8
1:
##FORMAT=<ID=DP,Number=1,Type=Integer,
Description="Read Depth">
FILTER = .
INFO
= AO=149;RO=21;TYPE=snp
8
= 1:170:21:788:149:5579:-5,0
Variant Call Format
##INFO=<ID=RO,Number=1,Type=Integer,Description="
Reference allele observation count, with partial
##INFO=<ID=AO,Number=A,Type=Integer,Description="
Alternate allele observations, with partial
##INFO=<ID=TYPE,Number=A,Type=String,Description="The
type of allele, either snp, mnp, ins, del, or
complex.">
Variant Call Format
##FORMAT=<ID=GT,Number=1,Type=String,Description="
Genotype">
##FORMAT=<ID=GQ,Number=1,Type=Float,Description="
Genotype Quality, the Phred-scaled marginal (or
##FORMAT=<ID=GL,Number=G,Type=Float,Description="
Genotype Likelihood, log10-scaled likelihoods of the
data given the called genotype for each possible
genotype generated from the reference and alternate
alleles given the sample ploidy">
##FORMAT=<ID=DP,Number=1,Type=Integer,Description="
Read Depth">
Variant Call Format
##FORMAT=<ID=RO,Number=1,Type=Integer,Description="
Reference allele observation count">
##FORMAT=<ID=QR,Number=1,Type=Integer,Description="
Sum of quality of the reference observations">
##FORMAT=<ID=AO,Number=A,Type=Integer,Description="
Alternate allele observation count">
##FORMAT=<ID=QA,Number=A,Type=Integer,Description="
Sum of quality of the alternate observations">
Variant Effect Prediction
● snpEff
● Variant Effect Predictor (EMBL)
● SIFT
VCF after Effect Prediction
#CHROM POS
ID
REF
ALT
QUAL
FILTER INFO
FORMAT 8
8_PB2
407
.
A
G
3935.83 .
AO=149;RO=21;TYPE=snp;EFF=SYNONYMOUS_CODING
(LOW|SILENT|gaA/gaG|E123|759|PB2||CODING|Tr_PB2|1|1) GT:DP:RO:QR:AO:QA:GL
1:170:21:788:149:5579:-5,0
CHROM
= 8_PB2
POS
= 407
ID
= .
REF
= A
ALT
= G
QUAL
= 3935.83
FILTER = .
INFO
= AO=149;RO=21;TYPE=snp;EFF=SYNONYMOUS_CODING
(LOW|SILENT|gaA/gaG|E123|759|PB2||CODING|Tr_PB2|1|1)
8
= 1:170:21:788:149:5579:-5,0
VCF after Effect Prediction
##INFO=<ID=TYPE,Number=A,Type=String,Description="The type of
allele, either snp, mnp, ins, del, or complex.">
##INFO=<ID=EFF,Number=.,Type=String,Description="Predicted
effects for this variant.Format: 'Effect ( Effect_Impact |
Functional_Class | Codon_Change | Amino_Acid_change |
Amino_Acid_length | Gene_Name | Transcript_BioType | Gene_Coding
| Transcript_ID | Exon | GenotypeNum [ | ERRORS | WARNINGS ] )'
">
INFO
= AO=149;RO=21;TYPE=snp;
EFF=SYNONYMOUS_CODING
(LOW|SILENT|gaA/gaG|E123|759|PB2||CODING|Tr_PB2|1|1)

Alignment - UC Davis Bioinformatics Core

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