Introduction:

In bioinformatics, BAM files are essential for storing aligned sequencing data, typically in the Binary Alignment/Map format. Sorting BAM files is crucial for various downstream applications, including variant calling, indexing, and visualization. A BAM file can be sorted either by query name (unsorted) or by genomic coordinates (sorted), with the latter being necessary for certain analyses.

Why Is It Important to Check if a BAM File is Sorted?

1. Efficient Access: Many tools, such as samtools and Picard, assume that BAM files are sorted by genomic coordinates. Without sorting, these tools may not function properly or efficiently.
2. Variant Calling: Most variant callers, like GATK, expect sorted BAM files to ensure correct processing of sequence alignments for variant discovery.
3. Indexing: To index a BAM file, it must be sorted by coordinates. An unsorted BAM file can cause indexing failures or incorrect outputs.
4. Visualizations: Genome browsers like IGV expect BAM files to be sorted to display alignments accurately.

How to Check If a BAM File is Sorted?

Method 1: Using samtools (Modern Approach)

Samtools is one of the most popular tools for handling BAM files. In recent versions, the command samtools stats provides an easy way to check if a BAM file is sorted.

1. Run samtools stats on Your BAM File:
   bash

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   samtools stats <file.bam> | grep "is sorted:"

2. Interpret the Output:
   • If the output is is sorted: 1, it means the BAM file is sorted by genomic coordinates.
   • If the output is is sorted: 0, the BAM file is not sorted.

   Example:

   bash

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   samtools stats myfile.bam | grep "is sorted:"
SN is sorted: 1


   This output indicates the BAM file is sorted.

Method 2: Inspecting the BAM Header

The BAM file header contains metadata, including sorting information. You can check this using samtools view:

1. Check BAM File Header:
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   samtools view -H <file.bam>

2. Look for the Sort Order (SO) Flag:
   • If the header contains SO:coordinate, the BAM file is sorted by coordinates.
   • If it contains SO:unsorted, the BAM file is not sorted.

   Example:

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   @HD VN:1.0 SO:coordinate


   This header line indicates that the BAM file is sorted by coordinates.

Method 3: Using samtools index

Samtools can also help identify whether a BAM file is sorted. If the BAM file is unsorted, samtools index might produce an error or a smaller index file. However, this method is not foolproof, as it can sometimes run without error on an unsorted file.

1. Index the BAM File:
   bash

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   samtools index <file.bam>

2. Check the Return Code:
   • If the file is unsorted, the command may produce a truncated index file, or you may receive an error like [bam_index_core] the alignment is not sorted.
   • If the BAM file is sorted, samtools index will run without errors and generate a correct index file.

   Example:

   bash

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   samtools index myfile.bam
echo $?

   • A return code of 0 indicates that samtools index has run successfully, suggesting the file is sorted.
   • A return code other than 0 may indicate the file is unsorted.

How to Sort a BAM File?

If you find that your BAM file is unsorted, you can sort it using samtools:

1. Sort the BAM File by Coordinates:
   bash

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   samtools sort <file.bam> -o <sorted_file.bam>

2. Verify the Sort: After sorting, you can verify that the BAM file is now sorted by checking its header or running the samtools stats command again.

Additional Considerations:

• MarkDuplicates: When using tools like MarkDuplicates, it’s crucial to set the assume_sorted=true option only if the BAM file is actually sorted. If you’re unsure, it’s always safest to run samtools sort first.
• samtools view: When working with SAM files, you can pipe the output of samtools view into a sorting command if necessary.

Unix or Perl Scripts for Automation:

You can automate the checking process with a simple shell script:

This script takes a BAM file as input, checks if it is sorted, and outputs the result.

Conclusion:

Ensuring that BAM files are sorted is crucial for the proper functioning of various bioinformatics tools. The most reliable way to check whether a BAM file is sorted is by using samtools stats. If necessary, you can sort the file using samtools sort. By automating these checks, you can streamline your bioinformatics workflow and avoid errors in downstream analyses.