Extracting reads from a BAM file that fall entirely within a given region. The process ensures you exclude reads overlapping but not contained within the specified region.

Step-by-Step Guide to Extract Reads Entirely Within a Region

1. Requirements

• Software:
  • samtools (for manipulating BAM files)
  • bedtools (for handling BED files)
  • bcftools (optional for variant analysis)
• Input files:
  • A sorted and indexed BAM file (.bam, .bai)
  • A region file in BED or GFF format (e.g., regions.bed).

2. Install Necessary Tools

• On a Unix/Linux system, use the following commands to install required tools:
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  sudo apt-get install samtools bedtools


  Alternatively, use conda:

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  conda install -c bioconda samtools bedtools


3. Prepare Input Files

• Ensure your BAM file is sorted and indexed:
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  samtools sort input.bam -o input_sorted.bam
samtools index input_sorted.bam

• Format the BED file to specify the regions:
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  chr10 1000 2000


  Save this as regions.bed.

4. Extract Reads Within Regions

• Use samtools view to extract reads overlapping the region:
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  samtools view -L regions.bed input_sorted.bam > overlapping_reads.sam

• Convert to BAM format and include the header for downstream compatibility:
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  samtools view -b -L regions.bed input_sorted.bam > overlapping_reads.bam


5. Filter Reads Entirely Within a Region

• Convert BAM to BED format for detailed filtering:
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  bedtools bamtobed -i overlapping_reads.bam > overlapping_reads.bed

• Use awk or bedtools to filter reads entirely within regions:
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  bedtools intersect -a overlapping_reads.bed -b regions.bed -f 1.0 > reads_within.bed

  • Explanation:
    • -f 1.0 ensures that reads are fully contained within the region.
    • The output file reads_within.bed contains filtered reads.

6. Convert Filtered BED Back to BAM

• Convert the filtered BED file back to BAM format:
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  bedtools bedtobam -i reads_within.bed -g genome_file > filtered_reads.bam


  Replace genome_file with your genome’s chromosome size file (e.g., genome.chrom.sizes).

7. Validate the Output

• View the extracted reads:
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  samtools view filtered_reads.bam | less -S

• Count the number of reads:
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  samtools view -c filtered_reads.bam

• Visualize the BAM file using tools like IGV or Tablet.

Script for Automation

You can automate the above steps using a shell script:

Advanced Tips

• If working with paired-end reads, ensure both ends meet the filtering criteria using samtools view -f 2.
• To handle multiple regions in the BED file, ensure all regions are formatted correctly.
• Use R with GenomicRanges for a flexible programming solution:
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  library(GenomicAlignments)
bam <- readGAlignments("input_sorted.bam")
region <- GRanges("chr10", IRanges(1000, 2000))
contained <- subsetByOverlaps(bam, region, type = "within")
export(contained, "filtered_reads.bam", format = "BAM")


This guide provides an effective workflow for extracting reads strictly within a specific region, suitable for bioinformatics beginners.