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Forum: Pacific Biosciences 01-13-2017, 03:15 AM
Replies: 1
Views: 2,450
Posted By reubennowell
PacBio ENA SRA submission without bas.h5 files?

Dear all,

I've been sent some PacBio data from a collaborator that we now wish to submit to ENA. I've read that the way to do this is to create a manifest file, pointing to the 3 bax.h5 files, 1...
Forum: Pacific Biosciences 10-12-2015, 05:16 AM
Replies: 4
Views: 3,580
Posted By reubennowell
Thanks for the replies both. @GenoMax, yes I...

Thanks for the replies both.

@GenoMax, yes I tried to import and received a "no SMRT cell data here" message from the Import SMRT Cells page. I agree this data appears to have been partly...
Forum: Pacific Biosciences 10-11-2015, 02:56 PM
Replies: 4
Views: 3,580
Posted By reubennowell
Import raw PacBio data from *.bax.h5 files

Dear all,

I've been sent some raw PacBio data from a collaborator which I'd like to import into SMRT Portal to generate filtered subreads and CCSs for scaffolding. However I'm struggling to...
Forum: Bioinformatics 05-14-2015, 07:21 AM
Replies: 6
Views: 6,857
Posted By reubennowell
I found another case where this was happening I...

I found another case where this was happening I thought I'd just add to the list for future reference.

My input fasta file was line wrapped, and there was a blank line between sequences. The...
Forum: Bioinformatics 10-30-2014, 11:41 AM
Replies: 12
Views: 8,575
Posted By reubennowell
I was having the same issues, and I think it's do...

I was having the same issues, and I think it's do with the version of Java you are running. BRIG was written in Java 1.6, I don't think it works with 1.7 (on my system anyway). On Ubuntu, try java...
Forum: Bioinformatics 07-17-2014, 08:32 AM
Replies: 1
Views: 1,788
Posted By reubennowell
Weird SNPs - Assembly Error or Mapping Error?

Hello all,

I need some help interpreting what I'm seeing in my assembly data.

I'm doing some variant calling in a small number of closely related bacterial genomes: I assembled each strain de...
Forum: Bioinformatics 06-12-2013, 02:56 AM
Replies: 4
Views: 3,757
Posted By reubennowell
Cheers Torst! Using CutAdapt with the...

Cheers Torst!

Using CutAdapt with the reverse complement to the forward read transposase sequence trims a significant proportion of the R2 reads and removes the position-specific Kmer enrichment...
Forum: Bioinformatics 06-06-2013, 03:31 AM
Replies: 4
Views: 3,757
Posted By reubennowell
Hi Torst, Thanks for the link to Nick...

Hi Torst,

Thanks for the link to Nick Loman's (excellent) blog - I think that is exactly what I'm seeing. Increasing the kmer length with the -k flag in FastQC revealed the enrichment of the...
Forum: Bioinformatics 05-30-2013, 09:07 AM
Replies: 4
Views: 3,757
Posted By reubennowell
FastQC Kmer content: spike in reverse read

Hello,

I'm using FastQC to check the quality of my 250bp PE Miseq reads prior to assembly, and am seeing a spike in the relative enrichment of the 5-mer 'TTATA' around the 160-169 bp region of the...
Forum: Bioinformatics 05-23-2013, 11:08 AM
Replies: 7
Views: 3,218
Posted By reubennowell
Thanks guys, Nope, not RNA-Seq - this is...

Thanks guys,

Nope, not RNA-Seq - this is bacterial genomic DNA. Mastal, can you remember what you did to account for it? Trim the first X bases off the 5' end? In my datasets, it seems to...
Forum: Bioinformatics 05-23-2013, 07:46 AM
Replies: 7
Views: 3,218
Posted By reubennowell
First 20bp of MiSeq reads are weird

Hello,

The first 20 bases of my MiSeq reads show abnormal %A, T, G and C, as evidenced by the 'per base sequence content' tab of the FastQC report (see the attached PNG). The per base GC content...
Forum: Bioinformatics 05-06-2013, 02:55 AM
Replies: 17
Views: 3,526
Posted By reubennowell
Yeah, there are zero N's in the scaffolds.fa file...

Yeah, there are zero N's in the scaffolds.fa file in my assemblies also :) I'm going to run SSPACE and GapFiller anyway to see if there is further improvement.

RE the small contigs (<500bp):...
Forum: Bioinformatics 05-03-2013, 02:50 AM
Replies: 17
Views: 3,526
Posted By reubennowell
Thanks! That worked a treat. And running it up...

Thanks! That worked a treat. And running it up to k=250 produced an assembly with the highest N50 I've managed to produce thus far (>60kb for a 6Mb bacterial genome), across a range of assemblers...
Forum: Bioinformatics 05-02-2013, 08:48 AM
Replies: 17
Views: 3,526
Posted By reubennowell
That's interesting - so had you run FLASH or...

That's interesting - so had you run FLASH or something similar also? Was the distribution of read lengths similar to what I have?

Also, idba_ud has a max kmer of <= 124. How did you recompile...
Forum: Bioinformatics 04-26-2013, 08:11 AM
Replies: 17
Views: 3,526
Posted By reubennowell
Hi Boetsie, well I'm running velvet with a kmer...

Hi Boetsie, well I'm running velvet with a kmer of around 127 currently, so quite large already. I tried upping it a bit, with no improvements in the assembly statistics. But there is considerable...
Forum: Bioinformatics 04-23-2013, 09:56 AM
Replies: 17
Views: 3,526
Posted By reubennowell
Thanks again Torst, unfortunately the sequencing...

Thanks again Torst, unfortunately the sequencing stage of our project is completed - we'll have to work with what we've got!

I have now tried using FLASH with more realistic input parameters (-m...
Forum: Bioinformatics 04-17-2013, 08:43 AM
Replies: 17
Views: 3,526
Posted By reubennowell
Hello and thanks for the advice Torst, ...

Hello and thanks for the advice Torst,

Firstly, yes, these are Pseudomonas genomes - well deduced! How did you guess??

So, just to clarify, the reads have been adapter-trimmed using CutAdapt...
Forum: Bioinformatics 04-16-2013, 07:43 AM
Replies: 17
Views: 3,526
Posted By reubennowell
How to set -max_coverage in velvetg

Hello,

I am assembling bacterial genomes (~6Mb) using 250 bp paired-end MiSeq data. I have tried a bunch of assemblers (idba_ud, mira, ray, SOAPdenovo, ABySS to name a few...), but am getting...
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