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
（也可以利用FLAG值含义解释工具：https://www.plob.org/2012/02/04/1697.html）
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):
| 1 | the read is paired in sequencing, no matter whether it is mapped in a pair |
| 1 | the read is mapped in a proper pair (depends on the protocol, normally inferred during alignment) |
| 0 | the query sequence itself is unmapped |
| 0 | the mate is unmapped |
| 0 | strand of the query (0 for forward; 1 for reverse strand) |
| 1 | strand of the mate |
| 0 | the read is the first read in a pair |
| 1 | the 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 http://www.hudsonalpha.org/gsl/software/bam2fastq.php
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
More information
http://samtools.sourceforge.net/
http://sourceforge.net/apps/mediawiki/samtools/index.php?title=SAM_FAQ
