80    ◾    Bioinformatics

samtools view \

-@ 4 \

-uS -o SRR769545_mem.bam SRR769545_mem.sam

The “-@” option specifies the number of threads, the “-u” option is to produce uncom-

pressed BAM output, “-S” is to ignore auto-detection of the input format, and “-o” specifies

the output file.

If you have already run the above command and the old SAM file is still there, you can

delete it with “rm SRR769545_mem.sam” command and then run the following to convert

the BAM file back to a SAM file:

samtools view \

-@ 4 -h \

-o SRR769545_mem.sam \

SRR769545_mem.bam

The “-h” option is to include header in SAM output.

2.4.1.2  Sorting Alignments

Sorted alignments are required for some applications such as variant calling and RNA-Seq.

Samtools can sort the alignments in a BAM file by coordinate order, by read name, or by

a TAG. By default, “samtools sort” will sort the alignment by the coordinate order, unless

“-n” or “-t TAG” option is used. If “-n” option is used, the command will sort the align-

ments by read name. If the “-t TAG” option is used, then the alignments will be sorted by

tag. Refer to SAM and BAM file formats discussed above to learn about the standard TAGs.

In the following, the BAM file will be sorted by coordinate order:

samtools sort \

-@ 4 \

-T mem.tmp.sort \

-o SRR769545_mem_sorted.bam \

SRR769545_mem.bam

This will create a new BAM file with sorted alignments. You can delete the unsorted BAM

file if you need to save some storage space.

2.4.1.3  Indexing BAM File

Before using a BAM file in any of the downstream analysis, it can be indexed to allow fast

random access to the alignments in the file. Thus, the alignment information will be pro-

cessed faster. We can use the “samtools index” command to index the above sorted BAM

file as follows:

samtools index SRR769545_mem_sorted.bam

This will generate the BAM index file “SRR769545_mem_sorted.bam.

bai”.