Mapping to Reference Talk

Mapping to a reference genome

Contents

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

The mapping process

Gettng 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.

Mapping is a vital step

NGS data - Challenges

• Big Data (massive scale 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

Mapping process considerations 1

• Different mappers depending on:

- Read length

- SNVs? Indels?

- DNA or RNA

- Single end or paired end?

- Should multiple hits be allowed?

So, which mapper to use?

Mapping process considerations 2

Mapping algorithms

• BLAST

- Allows comparing and searching amino-acid and DNA sequences in a database of sequences

- Uses a heuristic algorithm: cannot guarantee the optimal alignment

- Too slow for NGS

• Hash-based mappers

- High memory footprint

- Slow for NGS

• Burrows Wheeler Transform

- Very fast and low memory footprint

- Very sensitive to errors

• Hybrids

Mapping output

SAM format example (http://samtools.github.io/hts-specs/SAMv1.pdf)

SAM/BAM format: header section

SAM/BAM format: alignment section

SAM/BAM format: FLAG

e.g. 1059, what does it mean? http://broadinstitute.github.io/picard/explain-flags.html

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.

SAM/BAM format: CIGAR example

SAM/BAM format: optional tags

optags1.png

SAM/BAM format: option tags

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 by Heng Li

- Provide various utilities for manipulating alignments in the SAM format

- SAM to BAM conversion

- Sorting (by coordinates or query-name)

- Merging several files

- BAM index

• picard-tools (essentially a version of samtools for Java)
• Pysam (Python)
• Rsamtools (R/Bioconductor)

Tophat

• 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