7. Advanced usage

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# 7. Advanced usage

## 7.1. Mask all regions in a genome except for targeted capture regions.

Step 1. Add 500 bp up and downstream of each probe

    bedtools slop -i probes.bed -b 500 > probes.500bp.bed

Step 2. Get a BED file of all regions not covered by the probes (+500 bp up/down)

    bedtools complement -i probes.500bp.bed -g hg18.genome > probes.500bp.complement.bed

Step 3. Create a masked genome where all bases are masked except for the probes +500bp

    bedtools maskfasta -in hg18.fa -bed probes.500bp.complement.bed -fo \
    > hg18.probecomplement.masked.fa

## 7.2. Screening for novel SNPs.

Find all SNPs that are not in dbSnp and not in the latest 1000 genomes calls

    bedtools intersect -a snp.calls.bed -b dbSnp.bed -v | \
    bedtools intersect -a - -b 1KG.bed -v | \
    > snp.calls.novel.bed

## 7.3. Computing the coverage of features that align entirely within an interval.

By default, bedtools `coverage` counts any feature in A that overlaps B by >= 1 bp. If you want to require that a feature align entirely within B for it to be counted, you can first use intersectBed with the “-f 1.0” option.

    bedtools intersect -a features.bed -b windows.bed -f 1.0 | \
    bedtools coverage -a - -b windows.bed \
    > windows.bed.coverage

## 7.4. Computing the coverage of BAM alignments on exons.

One can combine `samtools` with `bedtools` to compute coverage directly from the BAM data by using `bamtobed`.

    bedtools bamtobed -i reads.bam | \
    bedtools coverage -a - -b exons.bed \
    > exons.bed.coverage

Take it a step further and require that coverage be from properly-paired reads.

    samtools view -uf 0x2 reads.bam | \
    coverageBed -abam - -b exons.bed \
    > exons.bed.proper.coverage

## 7.5. Computing coverage separately for each strand.

Use grep to only look at forward strand features (i.e. those that end in “+”).

    bedtools bamtobed -i reads.bam | \
    grep \+$  | \
    bedtools coverage -a - -b genes.bed \
    > genes.bed.forward.coverage

Use grep to only look at reverse strand features (i.e. those that end in “-”).

    bedtools bamtobed -i reads.bam | \
    grep \-$ | \
    bedtools coverage -a - -b genes.bed \
    > genes.bed.reverse.coverage

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