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The command for samtools to filter multiple alignment reads is as follows:

Samtools view-h-Q 1-F 4-F 256 big.sort.dedup.bam | grep-v XA:Z | grep-v SA:Z | samtools view-b-> big.sort.dedup.unique.bam

Where the parameters are:

-Q 1: filter out reads whose comparison mass is less than 1

-F 4: filter out unmatched reads

-F 256: filter out multiple reads compared to each other.

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