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Extracting Novel transcript sequence from RNA-seq alignment sahilsukla Introductions 0 08-09-2012 12:19 AM

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Old 11-13-2013, 01:47 PM   #1
FLYINGDOLPHIN
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Default extra sequence following transcript in RNA-seq ?

Hi,

I did a dual-seq with pathogen and host. I found more nucleotides after the total mRNA transcript(as annotation) from the pathogen. For each sequence that mapped to the pathogen transcript in the fastq file, the strings of nuceotides following the end of the transcript, however are very different. This role out the possibility that the annotation sequence is not intact.

Does anyone know whether this happens often? And why this would happen?

Thanks a lot!

Best,
F
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Old 11-13-2013, 03:19 PM   #2
gringer
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You can get this happening off the end of transcripts because the sequencer just sequences whatever it has available (if anything). For circularised DNA, this ends up being the index sequences that are attached to the ends of the reads. Have you done adapter clipping prior to mapping?
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Old 11-13-2013, 03:32 PM   #3
FLYINGDOLPHIN
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Hi Gringer,

Thanks for the quick reply. I got the data after they are removed off the index sequences. All the fragments that I get are of equal length. I just don't why there are still nucleotides after the end of the transcripts.
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Old 11-14-2013, 06:07 AM   #4
NicoBxl
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Could you post a picture of your alignment.
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Old 11-14-2013, 09:21 AM   #5
FLYINGDOLPHIN
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Hi,

I grep the pathogen sequence from the fastq file.

All the matched seuqence is 50bp.

Before the space is the end of my pathogen transcript. Any nucleotides following it, I don't know what they are. Could this be technical noise?

Thanks.
F
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Old 11-14-2013, 12:12 PM   #6
gringer
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Quote:
Originally Posted by FLYINGDOLPHIN View Post
Could this be technical noise?
If that's the case, then you should be able to tell by the quality scores in the FASTQ files. If it's substantially lower than the transcript sequence (e.g. 10ish), then it's probably noise due to the machine getting confused. It doesn't look like it's due to phasing alone, because then you'd expect to get more sequence that looks similar to the last few bases (e.g. the line that has '20' next to it).
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