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Old 01-19-2012, 01:54 AM   #1
Location: Germany

Join Date: Jun 2011
Posts: 54
Question reads mapping across scaffold boundaries


I am struggling with assembling a transcriptome with SE 454 reads. I used bwa-sw to map the reads and want to use cufflinks for the assembly. Eventually I get errors like these
>Error (GFaSeqGet): end coordinate (994) cannot be larger than sequence length 883
>Error (GFaSeqGet): subsequence cannot be larger than 472
>Error getting subseq for CUFF.18.1 (13..673)!
Here's what I think is happening:
I have about 12000 scaffolds, most of which are tiny. I have reads that map beyond the end of a scaffold, meaning that a part of the read is not aligned to the scaffold but "hangs over".

read -----------------
scaffold: |----------------------|

If I then try to extract the transcript with cufflinks, I get the error, because Cufflinks uses the reference genome to extract the exons instead of the assembled reads.

This now raised three questions for me:
1. Why do I have quite a number of reads mapping to tiny scaffolds. Assuming that these scaffolds are repetitive DNA or junk or whatever excluded them from being assembled in a bigger scaffold.
2. Is there a way to extract the transcripts with a different software?
3. Or should I try to exclude either all small scaffolds or all reads mapping beyond scaffold ends from my transcriptome assembly?
lukas1848 is offline   Reply With Quote
Old 03-21-2012, 05:03 PM   #2
Location: NJ

Join Date: Feb 2010
Posts: 54

I've got the same issue here... anyone found a solution yet?
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Old 03-28-2012, 08:26 AM   #3
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Location: Durham, NH

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of note, you can eliminate this error by NOT using the -b option in cufflinks.. Also, this bug is exposed in bowtie2 (, but not in bowtie1, at least I've never had it happen with B1..
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Old 01-30-2013, 10:15 AM   #4
Location: Atlanta

Join Date: Apr 2011
Posts: 32

Error (GFaSeqGet): subsequence cannot be larger than 16571
Error getting subseq for CUFF.42374.1 (2..16614)!

I am getting the same error....has anybody found a solution?? -b with or without does not work for me
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Old 01-30-2013, 12:00 PM   #5
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Location: San Diego

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Posts: 912

You could pad all your references with Ns so that nothing hangs off.
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Old 01-30-2013, 01:15 PM   #6
Location: Atlanta

Join Date: Apr 2011
Posts: 32

Forgive my ignorance...but how do I do pad those sequences?
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Old 05-30-2016, 11:13 PM   #7
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Location: UK

Join Date: Feb 2014
Posts: 206

Hi I have same problem. It seems that lots of people have this error.

I use the Ensembl genome(*dna.toplevel.fa) and GTF file.

If I ran cufflinks (even without -b genome.fa) but then run Cuffmerge with -s genome.fa, I got the error!

If I omitted -b genome.fa in both Cufflinks and Cuffdiff and meanwhile omit -s genome.fa in Cuffmerge, I didn't get the error! However, I don't know if it was accurate.
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