Thanks, that's great news!
On Nov 14, 2013 9:29 PM, "Wei Shi" <shi@wehi.edu.au> wrote:

> Hi Ryan,
>
> We have made changes to featureCounts to let it deal with those read pairs
> that having missing mates. featureCounts now also checks if reads from the
> same pair are adjacent to each other and if not it will automatically
> re-order the reads to make them ready for read counting. So you do not need
> to specify the PEReadsReordering option any more (it is now removed from
> the function) and you do not need to use the separate bam file which
> includes unmapped reads either.
>
> Changes have been committed to bioc devel and they should be available in
> a couple of days (Rsubread v1.13.6)
>
> Wei
>
> On Nov 11, 2013, at 12:23 PM, Ryan wrote:
>
> > Ah, I see now. I am using tophat, which outputs the unmapped reads to a
> separate bam file. I guess I should merge the two bam files and then
> featureCounts should work as I expect?
> >
> > On Sun Nov 10 16:59:32 2013, Wei Shi wrote:
> >> Dear Ryan,
> >>
> >> featureCounts requires that both reads from the same pair are present
> in the BAM/SAM file. But this doesn't seem to be the case for your data. It
> looks like unmapped reads were not included in your BAM files and therefore
> you got quite a few unpaired reads in your data. When counting in
> paired-end mode (isPairedEnd=TRUE) and read reordering being required
> (PEReadsReordering=TREU), featureCounts tries to reorder reads from the
> same pair to make them be adjacent to each other before performing read
> counting. If unpaired reads are encountered, featureCounts simply discards
> them.
> >>
> >> Simply sorting your bam files by name is not going to solve this
> problem because many reads are unpaired in your data. You may use an
> aligner that outputs all the reads in it mapping output (such as the align
> function in Rsubread) and then runs featureCounts on it.
> >>
> >> In the meantime, we are now trying to improve featureCounts to deal
> with this simulation. Will let you know when this is done.
> >>
> >> Also note that this problem only occurs for paired-end data, but not
> for single-end read data.
> >>
> >> Best regards,
> >> Wei
> >>
> >> On Nov 9, 2013, at 10:54 AM, Ryan C. Thompson wrote:
> >>
> >>> Hello,
> >>>
> >>> I am using the featureCounts function in Rsubread to count RNA-seq
> reads. Since my BAM files are coordinate-sorted paired-end, I am using
> "isPairedEnd=TRUE" and "PEReadsReordering=TRUE". When I run my script, I
> see the following in the output:
> >>>
> >>> Process /gpfs/home/rcthomps/Projects/cyno/tophat_results/R77_CN7314_P4
> ...
> >>>   Resort the input file ...
> >>>   2718714 unpaired reads were ignored in reordering.
> >>>   Total number of fragments is : 1653582
> >>>   Number of successfully assigned fragments is : 1529491 (92.5%)
> >>>   Running time : 3.39 minutes
> >>>
> >>> In particular, I am worried about the line "2718714 unpaired reads
> were ignored in reordering." The documentation for PEReadsReordering
> doesn't say anything about discarding unpaired reads, so I'm wondering
> whether this is intended behavior, and whether there is a way around this?
> If I sort my bam files by name so that pairs are adjacent in the file, will
> subread still discard the unpaired ones, or will it count all of them?
> >>>
> >>> -Ryan Thompson
> >>>
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