Skip to content

Latest commit

 

History

History
127 lines (97 loc) · 3.71 KB

SAM_BAM_queries.md

File metadata and controls

127 lines (97 loc) · 3.71 KB

After mapping the fastq file to the reference genome you will end up with a SAM or BAM alignment file.

  • SAM format: SAM stands for Sequence Alignment/Map format. A single SAM file can store mapped, unmapped, and even QC-failed reads from a sequencing run, and indexed to allow rapid access: this means that the raw sequencing data can be fully recapitulated from the SAM/BAM file.

  • BAM format: SAM is rarely helpful and really takes up too much space which is why we use only the BAM in principle. A BAM file (.bam) is the binary version of a SAM file (saving storage and faster manipulation)

  • SAM tools to explore SAM and BAM files

You can use samtools: a free software package for manipulating SAM/BAM files to manipulate your SAM/BAM files and extract different kind of information from them. Samtools provide utilities for:

  • Viewing and formatting
  • Extracting statistics
  • Indexing
  • Manipulating SAM/BAM files
  • Editing
Usage:  samtools <command> [options]

samtools offers many options, you can check them here

One of the most used tools since BAM files are often the input files needed for many different analysis programs.

samtools view

from SAM to BAM

samtools view -b file.sam > file.bam

or

samtools view -bT file.sam > file.bam
#if the header is absent from the SAM file

or

samtools view -bS file.sam > file.bam
#if the header is header information is available

from BAM to SAM

samtools view file.bam > file.sam

Use options –h and –H to deal with the header

  • samtools sort
#sorting a bam file
samtools sort file.bam –o file_sorted.bam

#converting SAM directly to a sorted BAM file
samtools view file.sam |samtools sort –o file_sorted.bam

SAM/BAM files can be sorted in multiple ways, e.g. by location of alignment on the chromosome, by read name, etc. Note that different alignment tools will output differently sorted SAM/BAM, and you might need differently sorted alignment files as input for different downstream analysis tools.

  • mapping statistics
samtools flagstat file.bam

does a full pass through the input file to calculate and print statistics such as: %reads mapped, % unmapped reads, % reads properly paired and Other information.

Many tools require a BAM Index file to more efficiently access reads in a BAM file.
To create a BAM index, you must first sort the BAM file to create a sorted.bam and then run samtools index with the sorted.bam as input This will create a file named sorted.bam.bai which contains the index.

samtools view  file.sam >file.bam
samtools sort file.bam -o file_sorted.bam
samtools index file_sorted.bam file_sorted.bai
  • Filtering out unmapped reads from BAM files
samtools view -h -F 4  file.bam > file_only_mapped.sam
# output back to BAM
samtools view -h -F 4 –b file.bam > file_only_mapped.bam

Extracting SAM entries mapping to a specific region

#index the bam file first
samtools index file.bam 

samtools view file.bam chr1:200000-500000
#all reads mapping on chr1 as another bam 
samtools view –b file.bam chr1 > file_chr1.bam
  • Computing the depth

Samtools allows computing the depth at each position using the depth option

samtools depth options file.bam

# –a allows to output all positions (including those with zero depth) 
samtools depth –a file.bam

#–q INT only count reads with base quality greater than INT
samtools depth –q int file.bam
  • Computing the coverage per region

Samtools allows computing the read depth per genomic region, as specified in the supplied BED file using Samtools bedcov

samtools bedcov options region.bed file.bam