== Summarizing, processing and quality control(QC) of SAM/BAM files ==

Many of these involve [http://samtools.sourceforge.net/samtools.shtml | samtools]

=== Convert, sort, and/or index ===


{{{
# Convert SAM to BAM:
samtools view -bS -o foo.bam foo.sam
}}}

{{{
# Convert BAM to SAM:
samtools view -h -o foo.sam foo.bam
}}}
{{{
# Sort BAM file (where ".bam" is added to "foo.sorted")
samtools sort foo.bam foo.sorted
}}}
{{{
# Index a sorted BAM file (which creates foo.sorted.bam.bai):
samtools index foo.sorted.bam
    
# Both foo.sorted.bam and foo.sorted.bam.bai are needed for visualization.
 }}}

All three steps (SAM=>BAM, sorting, and indexing) can be merged into one command.  See
{{{
/nfs/BaRC_Public/BaRC_code/Perl/SAM_to_BAM_sort_index/SAM_to_BAM_sort_index.pl
}}}

=== Process a BAM file into another BAM file ===

In many cases, there's no need to create an intermediate SAM file.  For example, to extract selected (mapped to chrM) reads:
{{{
samtools index accepted_hits.bam   # Required if you want to select a genome region (like chrM)
samtools view -h accepted_hits.bam chrM | samtools view -bS - > accepted_hits.chrM_only.bam
}}}
We need to keep the header to convert back to BAM (hence the '-h' with 'samtools view' and the '$1 ~ ...' with awk).

=== Count the number of mapped reads ===

{{{
samtools flagstat mapped_unmapped.bam
}}}

=== Count the number of mapped reads by chromosome ===

{{{
# Method 1 (all chromosomes)
# 1 - Index the BAM file: 
samtools index mapped_reads.bam
# 2 - Get index statistics (including the number of mapped reads in the third column: 
samtools idxstats mapped_reads.bam
}}}

{{{
# Method 2 (one chromosome at a time, for example, chr2)
# From SAM
awk -F"\t" '$3 == "chr2" {print $1}' mapped_reads.sam | sort -u | wc -l
# From BAM
samtools view mapped_reads.bam chr2 | cut -f 1 | sort -u | wc -l
}}}

=== Remove unmapped reads ===


{{{
samtools view -hS -F 4 mapped_unmapped.sam > mapped_only.sam
}}}


=== How many multiple/uniquely mapped reads are in a bam/sam file? ===

{{{
bam_stat.py -i mapped_reads.bam >& bam_stat.out.txt
}}}

=== View alignment with samtools ===

{{{
# -e: change identical bases to '='
samtools view -b accepted_hits.bam | samtools fillmd -e - /nfs/genomes/mouse_mm10_dec_11_no_random/fasta_whole_genome/mm10.fa | more
}}}


=== Get a list of multi-mapped reads, including the number of times each one was mapped ===

Tophat/bowtie mappers create the tag NH:i:XXX where XXX is the number of times the read has mapped.
{{{
bsub "samtools view accepted_hits.bam | grep -v NH:i:1 | perl -pe 's/AS.+(NH:i:\d+)/\$1/' | cut -f1,10,12 | perl -pe 's/NH:i://' | sort -u -k3,3nr > Multi-mapped.sorted.txt"
# Output format:
# read_ID<tab>read<tab>number times mapped
}}}

=== QC to get a (visual) summary of mapping statistics.  For eg. coverage/distribution of mapped reads across the genome or transcriptome. ===

==== RSeQC: RNA-Seq quality control package for getting mapping statistics (eg. unique/multi-mapped reads) ====
{{{
bam_stat.py -i myFile.bam
}}}

==== Picard: CollectRnaSeqMetrics.jar to find coverage across gene body for 5' or 3' bias ====
{{{
java -jar /usr/local/share/picard-tools/CollectRnaSeqMetrics.jar INPUT=accepted_hits.bam REF_FLAT=refFlat.txt STRAND_SPECIFICITY=NONE OUTPUT=Out_RnaSeqMetrics.txt REFERENCE_SEQUENCE=hg19.fa CHART_OUTPUT=Out_RnaSeqMetrics.pdf
}}}

==== QualiMap: can be used on DNA or RNA-Seq to get summary of mapping and coverage/distribution ====
{{{
# Graphical interface: enter 'qualimap' on the command line
# Command line:
unset DISPLAY  #needed for submitting to cluster
bsub "qualimap bamqc -bam myFile.bam -outdir output_qualimap"
#rnaseq qc
bsub "qualimap rnaseq -bam myFile.bam -gtf Homo_sapiens.GRCh37.72.canonical.gtf -outdir output_qualimap_rnaseq -protocol non-strand-specific"
}}}