Mapping to Reference Talk - Revision history

Rf at 23:19, 8 May 2017

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Rf at 23:16, 8 May 2017

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Rf at 22:30, 8 May 2017

2017-05-08T22:30:27Z

Rf: Created page with "Mapping to a reference genome = Contents = * Overview * Mapping process: Algorithms and tools * Mapping output: SAM/BAM specification Mapping to a reference genome U Trived..."

2017-05-07T12:22:54Z

Created page with "Mapping to a reference genome = Contents = * Overview * Mapping process: Algorithms and tools * Mapping output: SAM/BAM specification Mapping to a reference genome U Trived..."

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Mapping to a reference genome

= Contents =
* Overview
* Mapping process: Algorithms and tools
* Mapping output: SAM/BAM specification

Mapping to a reference genome

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= The mapping process =

Mapping to a reference genome

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= GeHng a Reference sequence =
* A reference is a consensus sequence, built up from high
quality sequencing samples. This can be genome or a
transcriptome.
* This should be in fasta format.

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= Mapping is a vital step =
Existing Reference Sequence

No Reference Sequence

Short Read Alignment
Variant Calling

De novo Assembly

De novo Transcriptome
Assembly

Gene Expression
Metagenomics
siRNA/microRNA
Analysis

Population Genomics

…

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= NGS data - Challenges =
* Massive Data:
* Illumina Hiseq 2500: 160GB in 2x150bp reads

* Natural variability: SNPs, indels, de novo
mutations, CNVs
* Sequencing errors
* RNA-seq: splice junctions to be considered
* Computing resources

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= Mapping process considerations =
Diﬀerent mappers depending on:
*
*
*
*
*

Read length
SNVs? Indels?
DNA or RNA
Single end or paired end?
Should mulFple hits be allowed?

Which mapper to use?
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= Mapping process considerations =

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= Mapping algorithms =
* BLAST

* Allows comparing and searching amino-acid and DNA
sequences in a database of sequences
* Uses a heurisFc algorithm: cannot guarantee the
opFmal alignment
* Too slow for NGS

* Based on Hashes

* High memory footprint
* Slow for NGS

* Burrows Wheeler Transform

* Very fast and low memory footprint
* Very sensiFve to errors

* Hybrids

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= Mapping output =
SAM/BAM format
http://samtools.github.io/hts-specs/SAMv1.pdf
Suppose we have the following alignment

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= Mapping output =
Corresponding SAM format will be:

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= SAM/BAM format: header section =

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= SAM/BAM format: alignment section =

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= SAM/BAM format: FLAG =

e.g. 1059, what does this mean?
http://broadinsFtute.github.io/picard/explain-ﬂags.html
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= SAM/BAM format: CIGAR =
* The CIGAR string is a sequence of base lengths with an associated
operation.
* Used to indicate things like which bases align (either a match/
mismatch) with the reference, are deleted from the reference, and
are insertions that are not in the reference.

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= SAM/BAM format: CIGAR example =

Alignment

CIGAR

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= SAM/BAM format: optional tags =

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= SAM/BAM format: option tags =

[Image]

= SAM/BAM format =
BAM format and BAM index
* BAM format
:- BAM format is the binary (compressed)
representation of a SAM file
:- A BAM file is smaller than its corresponding SAM file,
and can be read faster, but the content is the same

* BAM index
:- Indexing a bam allows to access the alignments from a specified region.
:- Required for alignment visualization soeware like IGV.

= SAM/BAM format: SAM parser =
* Samtools (http://samtools.sourceforge.net/)
:- Written in C (fast)
:- Provide various uFliFes for manipulating alignments in the SAM format
* SAM to BAM conversion
* Sorting (by coordinates or query-name)
* Merging several files
* BAM index

* Picard (Java)
* Pysam (Python)

= Tophat2 =

* First published 2009, up to that point, aligners behaved like so:
:- "whenever an RNA-Seq read spans an exon boundary, part of the read will not map contiguously to the reference, which causes the mapping procedure to fail for that read."
* Splicing-aware, finds annotated '''and''' novel junctions
* Reaching end-0f-life ... handing over to HISAT2

@@ Line 113: / Line 113: @@
 * picard-tools (essentially a version of samtools for Java)
 * Pysam (Python)
-* Rsamtools (R)
+* Rsamtools (R/Bioconductor)
 = Tophat =

@@ Line 5: / Line 5: @@
 * Mapping process: Algorithms and tools
 * Mapping output: SAM/BAM specification
 = The mapping process =
-Mapping to a reference genome
+[[File:mapproc.png]]
-= GeHng a Reference sequence =
+= Gettng a Reference sequence =
-* A reference is a consensus sequence, built up from high
+* A reference is a consensus sequence, built up from high quality sequencing samples. This can be genome or a transcriptome.
 * This should be in fasta format.
-Mapping to a reference genome
+[[File:hist.png]]
 = Mapping is a vital step =
+[[File:vital.png]]
 = NGS data - Challenges =
@@ Line 88: / Line 48: @@
 DNA or RNA
 Single end or paired end?
-Should mulFple hits be allowed?
+Should multiple hits be allowed?
 Which mapper to use?
@@ Line 110: / Line 70: @@
 * Allows comparing and searching amino-acid and DNA
 sequences in a database of sequences
-* Uses a heurisFc algorithm: cannot guarantee the
+* Uses a heuristic algorithm: cannot guarantee the
-opFmal alignment
+optimal alignment
 * Too slow for NGS
@@ Line 122: / Line 82: @@
 * Very fast and low memory footprint
-* Very sensiFve to errors
+* Very sensitive to errors
 * Hybrids
@@ Line 171: / Line 131: @@
 e.g. 1059, what does this mean?
-http://broadinsFtute.github.io/picard/explain-ﬂags.html
+http://broadinstitute.github.io/picard/explain-ﬂags.html
 Mapping to a reference genome
@@ Line 230: / Line 190: @@
 * Samtools (http://samtools.sourceforge.net/)
 :- Written in C (fast)
-:- Provide various uFliFes for manipulating alignments in the SAM format
+:- Provide various utilities for manipulating alignments in the SAM format
 * SAM to BAM conversion
 * Sorting (by coordinates or query-name)