[mira_talk] Re: Filtering out crappy sequence
- From: John Nash <john.he.nash@xxxxxxxxx>
- To: mira_talk@xxxxxxxxxxxxx
- Date: Wed, 4 Apr 2012 16:04:00 -0400
On 2012-04-04, at 3:53 PM, Peter Cock wrote:
> On Wed, Apr 4, 2012 at 8:45 PM, John Nash <john.he.nash@xxxxxxxxx> wrote:
>> On 2012-04-04, at 3:27 PM, Peter Cock wrote:
>>
>>> What were the failing sequences? Perhaps there is something we
>>> can suggest after seeing them and in what way they are bad.
>>>
>>
>> Hi Peter,
>>
>> I thought the same thing but the sequence looks like this (in fastq format):
>>
>> @HK6K99I01A94O4
>>
>>
>> +
>> !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
>> !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
>> !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
>> !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
>> !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
>> !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
>> !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
>>
>> I thought that 454 was configured not to output this sort of thing…
>
> That is very strange - did you remove the sequence or was it
> missing? If it was missing it was not valid FASTQ.
The sequence was directly converted from four sff files (which I got today) to
fastq using
sff_extract -Q -s PROJ_in.454.fastq -x PROJ_traceinfo_in.454.xml sff1 sff2
etc...
I didn't do any other manipulations.
>
> Also the quality string is odd, an exclamation mark is ASCII
> 33 which would be PHRED 1 on the Sanger encoding. This
Exactly
> suggests it is either a very bad read which should have
> failed QC and never made it to your SFF file, or something
> has gone very wrong in the SFF to FASTQ conversion.
>
Again, exactly. I was quite surprised by this.
> Which version of sff_extract did you use?
It was downloaded from the MIRA sourceforge repository in February 2012, and
'ls' tells me:
-rwxr-xr-x. 1 jnash jnash 52453 Sep 9 2011 sff_extract
>
> It would be worth testing the SFF file in other tools (e.g. the
> Roche applications or Biopython) to isolate the problem.
> If all the problem reads show this pattern (all their read
> qualities are PHRED 1) that should be easy to filter out.
I was just downloading some other converters to do that as your email came in :)
Thanks
John
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