A quick question: is there any way to extract uniquely aligned single-end or paired-end reads using Rsamtools? I read carefully about the documentation and did find a clear clue. IsSecondaryAlignment=T tag does not exclude a read which had aligned to multiple locations we designated as primary at other locations, correct? if so, then this won't serve for screening purpose.

Thanks for help

How would one do this with other tools? It seems like either the aligner inserts a unique tag (then use tagFilter = in ScanBamParam() or that one uses a heuristic based on properties of the aligner (mapqFilter =) or one sorts the bam file by qname (sortBam(..., byQname = TRUE) and eliminates duplicates (probably using filterBam() with a yieldSize to manage memory and a filter that looks for non-duplicate records).

If one were interested only in the primary alignment of possibly multiply aligned reads, then ScanBamParam(flag = scanBamFlag(isSecondaryAlignment = FALSE)) would do that, especially in conjunction with mapqFilter=.