Mapping to Reference Talk

Mapping to a reference genome

Contents

• Overview
• Mapping process: Algorithms and tools
• Mapping output: SAM/BAM specification

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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? Mapping to a reference genome

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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 Mapping to a reference genome

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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