smart-seq2 stranded

Hi Developers,

Below is an excerpt of logic SmartSeq2:

class SmartSeq2(Logic):
    """SmartSeq2 logic
    """

    def __init__(self) -> None:
        self.name = "SmartSeq2"

    @property
    def layers(self) -> List[str]:  # This should be overridden if a different set of layers is desired
        return ["spliced", "unspliced", "ambiguous", "spanning"]

    @property
    def stranded(self) -> bool:
        return False

I wonder if it true that SmartSeq2 will treat reads as non-stranded, even for stranded data? Thanks.

[luyoutao]

Thanks to your answer. That explains my observation that, when I tried to feed velocyto (with SmartSeq2 logic) a bam file with only R1 or R2, I got half number of counts as the entire bam file.

This behavior seems unexpected to me, because mainstream paired-end quantification tools avoid such double counting scheme. Here is an example from HTSeq document (http://htseq.readthedocs.io/en/master/count.html):

For paired-end data, does htseq-count count reads or read pairs? Read pairs. The script is designed to count “units of evidence” for gene expression. If both mates map to the same gene, this still only shows that one cDNA fragment originated from that gene. Hence, it should be counted only once.

[luyoutao]

Thanks again for your confirmation.

Sure. Bam file has read pair (among others) information encoded in “flag” (https://broadinstitute.github.io/picard/explain-flags.html): 0x40 indicates R1 and 0x80 R2. In terms of paired-end quantification logic, there are two widely used packages HTSeq (https://github.com/simon-anders/htseq/tree/master) and FeatureCount (http://subread.sourceforge.net/) you might refer to. Essentially, both package assigns a count to a gene, if one or both mates are aligned.