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Converting a SAM file to a BAM file

First, if you use the Unix command

head test.sam

The first 10 lines on your terminal after typing "head test.sam", should be lines starting with an "@" sign, which is an indicator for a header line. If you don't see lines starting with the "@" sign, the header information is most likely missing.

If the header information is absent from the SAM file use the command below, where reference.fa is the reference fasta file used to map the reads:

samtools view -bT reference.fa test.sam > test.bam

If the header information is available:

samtools view -bS test.sam > test.bam

Sorting a BAM file

samtools sort test.bam test_sorted

Creating a BAM index file

samtools index test_sorted.bam test_sorted.bai

Converting a BAM file to a SAM file

Note: remember to use -h to ensure the converted SAM file contains the header information. Generally, I suggest storing only sorted BAM files as they use much less disk space and are faster to process.

samtools view -h NA06984.chrom16.ILLUMINA.bwa.CEU.low_coverage.20100517.bam > NA06984.chrom16.ILLUMINA.bwa.CEU.low_coverage.20100517.sam

Simple stats

samtools flagstat NA06984.chrom16.ILLUMINA.bwa.CEU.low_coverage.20100517.bam

10182494 in total
0 QC failure
223627 duplicates
9861117 mapped (96.84%)
10095646 paired in sequencing
5049066 read1
5046580 read2
8174084 properly paired (80.97%)
9452892 with itself and mate mapped
321377 singletons (3.18%)
215316 with mate mapped to a different chr
126768 with mate mapped to a different chr (mapQ>=5)
For more statistics of SAM or BAM files have a look at the SAMStat program.

Interpreting the BAM flags


Here are some common BAM flags:

163: 10100011 in binary

147: 10010011 in binary

99: 1100011 in binary

83: 1010011 in binary

Interpretation of 10100011 (reading the binary from left to right):

1the read is paired in sequencing, no matter whether it is mapped in a pair1the read is mapped in a proper pair (depends on the protocol, normally inferred during alignment)0the query sequence itself is unmapped0the mate is unmapped0strand of the query (0 for forward; 1 for reverse strand)1strand of the mate0the read is the first read in a pair1the read is the second read in a pair

163 second read of a pair on the positive strand with negative strand mate

147 second read of a pair on the negative strand with positive strand mate

99 first read of a pair on the forward strand with negative strand mate

83 first read of a pair on the reverse strand with positive strand mate

Extracting only the first read from paired end BAM files

samtools view -h -f 0x0040 test.bam > test_first_pair.sam

0x0040 is hexadecimal for 64 (i.e. 16 * 4), which is binary for 1000000, corresponding to the read in the first read pair.

Filtering out unmapped reads in BAM files

samtools view -h -F 4 blah.bam > blah_only_mapped.sam

Creating FASTQ files from a BAM file

I found this great tool at

For example to extract ONLY unaligned from a bam file:

bam2fastq -o blah_unaligned.fastq --no-aligned blah.bam

To extract ONLY aligned reads from a bam file:

bam2fastq -o blah_aligned.fastq --no-unaligned blah.bam

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