Re: [Samtools-devel] Bit 0x4 and next segment

[Peter C. <p.j...@go...>]

On Thu, Feb 21, 2013 at 5:13 PM, James Bonfield <jk...@sa...> wrote:
> On Thu, Feb 21, 2013 at 02:47:06PM +0000, James Bonfield wrote:
>>   "Bit 0x4 is the only reliable place to tell whether the segment is
>>   unmapped."
>>
>>   ...
>>
>>   "If 0x4 is set, no assumptions may be mae about [...] the bit 0x20
>>   of the next segment in the template".
>
> While on the topic of this, I see it also says flag 0x10 is undefined
> if 0x04 is set.
>
> This has dire consequences for bam2fastq convertors as any unmapped
> reads cannot be converted back to fastq without making a decision one
> way or the other as to whether 0x10 (reverse comp) is valid.
>
> I see that bwa occasionally outputs nonsensical cigar strings and
> flags due to issues with mapping across two references. In these cases
> does it also set 0x10 if it reverse complemented the sequence?
>
> I believe we should tighten up the spec and state explicitly that flag
> 0x10 is valid and correct for unmapped reads. Ie if it states the
> sequence is complemented, then it is.
>
> James

Hi James

Why is there a problem in the spec?

If you have an unmapped read, it cannot be marked as reverse
complemented (under the current SAM/BAM FLAG definition),
therefore it must be stored in the original direction.

Any code storing a reverse complemented SEQ and QUAL but
setting the 0x10 bit for an unmapped read (0x4 set) is broken.

(Or is there a strong case to support this currently invalid
usage that is currently escaping me?)

Peter