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Forum: Bioinformatics 06-08-2020, 11:40 AM
Replies: 1
Views: 788
Posted By drdna
GATK calling false SNPs in duplicated regions

Is there a way to prevent GATK from calling SNPs in regions containing sequences that are duplicated in the reference genome? I'm having a problem with it calling false SNPs based on alignments of...
Forum: Bioinformatics 01-02-2020, 11:05 AM
Replies: 0
Views: 789
Posted By drdna
samtools mpileup bug

It seems to me that the samtools indeed calling function is very buggy. Recently called indels on a dataset where all indels were reported with massive forward strand bias. Two output examples are...
Forum: Oxford Nanopore 12-16-2019, 09:50 AM
Replies: 2
Views: 2,413
Posted By drdna
Expired AMPURE XP

Does anyone have any experience as to what happens when AMPURE XP reagent goes bad? We have been using it routinely to purify high molecular weight DNA for Nanopore sequencing but also of a sudden,...
Forum: Sample Prep / Library Generation 02-05-2019, 10:59 AM
Replies: 3
Views: 1,445
Posted By drdna
How are you measuring ligation?

How are you measuring ligation?
Forum: Bioinformatics 02-04-2019, 06:35 PM
Replies: 0
Views: 726
Posted By drdna
No LastGraph file from Velvet

Using VelvetOptimiser v2.2.6 to run a velveth, velvetg (v1.2.10) optimization procedure. Problem is that velvetg does not produce a LastGraph file. Instead the output directory contains just...
Forum: Bioinformatics 04-02-2018, 05:59 PM
Replies: 218
Views: 146,455
Posted By drdna
duplicated reference genomes

I don't understand why the genomeGenerate mode is creating a duplicated (concatenated) reference. This is resulting in at least two identical alignments for every read:

Command issued:

STAR...
Forum: Bioinformatics 11-11-2015, 05:53 AM
Replies: 1
Views: 2,035
Posted By drdna
DESeq: condition A vs condition B

How does DESeq which columns to assign to condition A and which to assign to column B? I previously ran an analysis in which column A corresponded to samples from males and column b corresponded to...
Forum: Bioinformatics 10-25-2015, 10:21 AM
Replies: 2
Views: 1,596
Posted By drdna
Bismark_methylation_extractor problem

I am getting the following error running bismark_methylation_extractor:

bismark_methylation_extractor --bedGraph --counts SS116_S1_L001_R1_001.fastq.gz_bismark_bt2_pe.bam
.......
Failed to...
Forum: Sample Prep / Library Generation 07-20-2015, 07:22 AM
Replies: 5
Views: 1,232
Posted By drdna
No, that's the original image that is also...

No, that's the original image that is also inaccurate for a different reason - see original post. It seems that the Illumina folks did not have anyone who understands the technology check the...
Forum: Sample Prep / Library Generation 07-20-2015, 06:59 AM
Replies: 5
Views: 1,232
Posted By drdna
This Illumina figure is also incorrect. As per my...

This Illumina figure is also incorrect. As per my understanding, it is not possible to generate double stranded molecules with free ends!

http://128.163.192.197/Illumina_mistake2.jpg

from the...
Forum: Sample Prep / Library Generation 07-19-2015, 07:20 PM
Replies: 5
Views: 1,232
Posted By drdna
Are the Illumina diagrams correct?

Is it true that single-stranded DNA libraries are initially BOUND directly to the flow cell as suggested in Illumina's diagrams (see panel b in attached figure). I always assumed they simply...
Forum: RNA Sequencing 07-14-2015, 08:53 AM
Replies: 6
Views: 2,711
Posted By drdna
The official header for that column is "score." ...

The official header for that column is "score." It only makes sense that this represents coverage. One could always run samtools mpileup on the relevant .bam file to check.
Forum: RNA Sequencing 07-14-2015, 08:39 AM
Replies: 6
Views: 2,711
Posted By drdna
Nope, it's read coverage. Column two indicates...

Nope, it's read coverage. Column two indicates position of first deleted base, column three indicates position of first retained base on other side of deletion.
Forum: RNA Sequencing 07-12-2015, 07:58 PM
Replies: 6
Views: 2,711
Posted By drdna
Depth of coverage

Depth of coverage
Forum: Bioinformatics 07-06-2015, 05:02 AM
Replies: 4
Views: 1,526
Posted By drdna
I am a seasoned bioinformatician with over 15 yrs...

I am a seasoned bioinformatician with over 15 yrs experience and yet running these programs is ridiculously frustrating. kmergenie works fine for kmer discovery but the suggested cov_cutoff causes...
Forum: Bioinformatics 07-05-2015, 08:51 AM
Replies: 4
Views: 1,526
Posted By drdna
After adjusting maxkmerlength, VelvetOptimiser...

After adjusting maxkmerlength, VelvetOptimiser selected a kmer size of 131. Problem is, this was decided based on an assembly that had two nodes of 195 and 285 bp and are supposed to represent a 34...
Forum: Bioinformatics 07-05-2015, 05:28 AM
Replies: 4
Views: 1,526
Posted By drdna
VelvetOptimiser failed assembly

First run through assembly of a mitochondrial genome sequenced with MiSeq (2 x 250) gave a pretty poor assembly with Kmers 25 through 31 (results shown for K31 - the best of the four). For...
Forum: Bioinformatics 12-24-2014, 07:30 AM
Replies: 189
Views: 74,543
Posted By drdna
Thanks for the insights. BaseSpace does a good...

Thanks for the insights. BaseSpace does a good job of adaptor trimming, demultiplexing. It's probably more efficient to just run the reads through a script to pair them up properly before downstream...
Forum: Bioinformatics 12-24-2014, 07:27 AM
Replies: 189
Views: 74,543
Posted By drdna
That's probably it - we use BaseSpace. My guess...

That's probably it - we use BaseSpace. My guess is that BaseSpace is filtering out pairs with poor quality. I'll have to look into that.
Forum: Bioinformatics 12-24-2014, 07:15 AM
Replies: 189
Views: 74,543
Posted By drdna
We have a MiSeq which gives us scads of high...

We have a MiSeq which gives us scads of high quality data but almost never gives us perfectly paired reads. I'll have to check with our tech to see if she sets any kind of paired data flag. Where...
Forum: Bioinformatics 12-24-2014, 06:59 AM
Replies: 189
Views: 74,543
Posted By drdna
This was the problem. Interesting, there was, to...

This was the problem. Interesting, there was, to my knowledge, no stipulation in the original publication, or in the online manual, that the program requires input files with perfectly paired reads....
Forum: Bioinformatics 12-23-2014, 08:09 PM
Replies: 189
Views: 74,543
Posted By drdna
Trimmomatic is not working correctly in paired...

Trimmomatic is not working correctly in paired end mode:

Read names in output files are not in the correct order. Correct phase was lost at read #27 and there are additional phase changes...
Forum: Bioinformatics 12-23-2014, 07:01 PM
Replies: 0
Views: 829
Posted By drdna
Tophat2 output fields query

Can someone please explain how the RNEXT and PNEXT fields in the sam/bam filea might be used? I can't see their utility.
Forum: Bioinformatics 12-13-2014, 01:55 PM
Replies: 7
Views: 1,617
Posted By drdna
Oops, I meant: samtools view -bSh File.sam >...

Oops, I meant:

samtools view -bSh File.sam > File.bam
Forum: Bioinformatics 12-13-2014, 12:52 PM
Replies: 7
Views: 1,617
Posted By drdna
It works if you force inclusion of the header...

It works if you force inclusion of the header when converting sam to bam:


samtools view -bSH File.sam > File.bam


Thanks for the heads up about the header blancha.
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