Counting the number of reads in a BAM file

The output from short read aligners like Bowtie and BWA is commonly stored in SAM/BAM format. When presented with one of these files a common first task is to calculate the total number of alignments (reads) captured in the file. In this post I show some examples for finding the total number of reads using samtools and directly from Java code. For the examples below, I use the HG00173.chrom11 BAM file from the 1000 genomes project which can be downloaded here.

First, we look at using the samtools command directly. One way to get the total number of alignments is to simply dump the entire SAM file and tell samtools to count instead of print (-c option):

$ samtools view -c HG00173.chrom11.ILLUMINA.bwa.FIN.low_coverage.20111114.bam
5218322

If we’re only interested in counting the total number of mapped reads we can add the -F 4 flag. Alternativley, we can count only the unmapped reads with -f 4:

# Mapped reads only
$ samtools view -c -F 4 HG00173.chrom11.ILLUMINA.bwa.FIN.low_coverage.20111114.bam
5068340

# Unmapped reads only
$ samtools view -c -f 4 HG00173.chrom11.ILLUMINA.bwa.FIN.low_coverage.20111114.bam
149982

To understand how this works we first need to inspect the SAM format. The SAM format includes a bitwise FLAG field described here. The -f/-F options to the samtools command allow us to query based on the presense/absence of bits in the FLAG field. So -f 4 only output alignments that are unmapped (flag 0x0004 is set) and -F 4 only output alignments that are not unmapped (i.e. flag 0x0004 is not set), hence these would only include mapped alignments.

An example for paired end reads you could do the following. To count the number of reads having both itself and it’s mate mapped:

$ samtools view -c -f 1 -F 12 HG00173.chrom11.ILLUMINA.bwa.FIN.low_coverage.20111114.bam
4906035

The -f 1 switch only includes reads that are paired in sequencing and -F 12 only includes reads that are not unmapped (flag 0x0004 is not set) and where the mate is not unmapped (flag 0x0008 is not set). Here we add 0x0004 + 0x0008 = 12 and use the -F (bits not set), meaning you want to include all reads where neither flag 0x0004 or 0x0008 is set. For help understanding the values for the SAM FLAG field there’s a handy web tool here.

There’s also a nice command included in samtools called flagstat which computes various summary statistics. However, I wasn’t able to find much documentation describing the output and it’s not mentioned anywhere in the man page. This post examines the C code for the flagstat command which provides some insight into the output.

$ samtools flagstat HG00173.chrom11.ILLUMINA.bwa.FIN.low_coverage.20111114.bam
5218322 + 0 in total (QC-passed reads + QC-failed reads)
273531 + 0 duplicates
5068340 + 0 mapped (97.13%:-nan%)
5205999 + 0 paired in sequencing
2603248 + 0 read1
2602751 + 0 read2
4881994 + 0 properly paired (93.78%:-nan%)
4906035 + 0 with itself and mate mapped
149982 + 0 singletons (2.88%:-nan%)
19869 + 0 with mate mapped to a different chr
15271 + 0 with mate mapped to a different chr (mapQ>=5)

The above shows a few simple examples using the samtools command but what if you wanted to count the total number of reads in code? I’ve been using the excellent Picard Java library as of late and haven’t found a simple way to do this via the API. I was looking for a fast way to compute this without having to scan the entire BAM file each time. Would love to see this added as a public function to the BAMIndexMetaData object or similar. Here’s a function I wrote to calcuate the total mapped reads from a BAM file. This makes use of the BAM index for speed and obviously requires you to first index your BAM file:

public int getTotalReadCount(SAMFileReader sam) {
    int count = 0;

    AbstractBAMFileIndex index = (AbstractBAMFileIndex) sam.getIndex();
    int nRefs = index.getNumberOfReferences();
    for (int i = 0; i < nRefs; i++) {
        BAMIndexMetaData meta = index.getMetaData(i);
        count += meta.getAlignedRecordCount();
    }

    return count;
}

This uses the BAMIndex to loop through each reference and sum the total mapped reads. A complete working example is included below:

import java.io.File;

import net.sf.samtools.AbstractBAMFileIndex;
import net.sf.samtools.BAMIndexMetaData;
import net.sf.samtools.SAMFileReader;

public class CountMapped {

    public static void main(String[] args) {
        File bamFile = new File(args[0]);

        SAMFileReader sam = new SAMFileReader(bamFile, 
                                 new File(bamFile.getAbsolutePath() + ".bai"));

        AbstractBAMFileIndex index = (AbstractBAMFileIndex) sam.getIndex();

        int count = 0;
        for (int i = 0; i < index.getNumberOfReferences(); i++) {
            BAMIndexMetaData meta = index.getMetaData(i);
            count += meta.getAlignedRecordCount();
        }

        System.out.println("Total mapped reads: " + count);
    }

}

Requires the Picard Java library. To compile/run:

$ javac -cp samtools.jar CountMapped.java
$ java -cp samtools.jar:. CountMapped HG00173.chrom11.ILLUMINA.bwa.FIN.low_coverage.20111114.bam
Total mapped reads: 5068340