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Forum: Bioinformatics 03-06-2016, 03:28 AM
Replies: 3
Views: 2,663
Posted By Studentlost
Exclamation Thank you for your reply! Just to make sure I...

Thank you for your reply! Just to make sure I follow, the library is basically the sample name so long as it's a single sample and only technical replicates, correct?

And the read group id is just...
Forum: Bioinformatics 03-05-2016, 07:59 PM
Replies: 3
Views: 2,663
Posted By Studentlost
Exclamation Confused about RG ID LB even after reading all posts/GATK best practices

I am getting conflicting information on how to assign RG ID, LB, PU, SM for an exome analysis I am working with.

Can someone just clarify for me how I should assign the RG please?

If you don't...
Forum: Bioinformatics 02-27-2016, 04:47 PM
Replies: 0
Views: 1,823
Posted By Studentlost
RNA-SEQ Quantification Pros/Cons using different features

I'm working with two replicates each for a two group pairwise comparison of single-end RNA Seq data derived from Illumina TruSeq v2, with fragments of ~50 bp.

I've aligned all samples using STAR...
Forum: Bioinformatics 02-21-2016, 06:04 PM
Replies: 1
Views: 1,762
Posted By Studentlost
STAR Alignment parameters for varying complexity in library

I'm working with TruSeq data for the mouse genome, with one of the groups having a low complexity library, and the other two having a high complexity library. The reads are single end, 50 bp.

What...
Forum: RNA Sequencing 12-16-2015, 01:59 PM
Replies: 1
Views: 1,041
Posted By Studentlost
Looking for gene markers unique across all contrasts

I have 8 groups with 3 replicates each. I've been working with edgeR, and doing pair-wise comparisons for the different groups. However, I'm interested in seeing which genes are unique markers across...
Forum: RNA Sequencing 01-27-2015, 07:14 PM
Replies: 0
Views: 1,126
Posted By Studentlost
Strand Rule Problem via rseqc in single end reads

Built an rna-seq pipeline for paired end reads.

Got single end data and had to revise.

Only problem I have now is the rseqc infer_Experiment strand_rule.

I am trying to run bam2big.py and...
Forum: RNA Sequencing 01-12-2015, 02:33 PM
Replies: 1
Views: 1,351
Posted By Studentlost
Parameters to consider for aligning long reads (150bp) in Tophat

Tophat's defaults sets: --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2.

This gave me about 4% alignment for mm9 mouse data.

I tried up to --read-mismatches 30 --read-gap-length 30...
Forum: RNA Sequencing 10-30-2014, 07:23 PM
Replies: 1
Views: 2,263
Posted By Studentlost
RSeqC Infer_Experiment Output Library Type?

Hey,

I'm trying to verify the strand reads for my ranseq_data using RSeQC's infer_experiment script.

I'm a bit confused by the documentation (found here:...
Forum: RNA Sequencing 10-30-2014, 07:20 PM
Replies: 34
Views: 5,489
Posted By Studentlost
I'm going to align with Star and I'll dump the...

I'm going to align with Star and I'll dump the alignment information for Bowtie2, Tophat2, and Star on here once that's finished this weekend so you guys can give me some insight.

Thanks again for...
Forum: RNA Sequencing 10-30-2014, 12:51 PM
Replies: 34
Views: 5,489
Posted By Studentlost
Sorry the SNAP in post 21 was a typo, I meant...

Sorry the SNAP in post 21 was a typo, I meant GSNAP. I haven't run it on GSNAP yet. I used Bowtie2 when I got the 46% alignment.

I am getting my alignment information via the align_summary.txt...
Forum: RNA Sequencing 10-28-2014, 09:27 PM
Replies: 34
Views: 5,489
Posted By Studentlost
If I knew the size of the fragments selected and...

If I knew the size of the fragments selected and sequenced, how would I implement that into my tophat code? Could you give me an example?
Forum: RNA Sequencing 10-28-2014, 08:30 PM
Replies: 34
Views: 5,489
Posted By Studentlost
I tried many variations. They all gave me the...

I tried many variations. They all gave me the same results. I've ran it with and without a library type (first strand and unstranded), with and without a reference transcriptome index, with and...
Forum: RNA Sequencing 10-28-2014, 07:55 PM
Replies: 34
Views: 5,489
Posted By Studentlost
I used bowtie2 (since tophat uses bowtie2 for...

I used bowtie2 (since tophat uses bowtie2 for alignment) as a test to see what was going on. I shouldn't be getting 0.4% alignment with tophat and 46% alignment with bowtie2. That's astronomically...
Forum: RNA Sequencing 10-28-2014, 06:33 PM
Replies: 34
Views: 5,489
Posted By Studentlost
The reads are of mRNA sequenced from single cells...

The reads are of mRNA sequenced from single cells of primates. I'm not sure how it was run, my knowledge starts off at the point of raw reads given to me. The genome is the Mmul_1 build by Ensembl,...
Forum: RNA Sequencing 10-28-2014, 06:14 PM
Replies: 34
Views: 5,489
Posted By Studentlost
I'm not sure I follow. Here is my...

I'm not sure I follow.

Here is my prep_reads.info file for one of my samples. This one is 396,555 base pairs:
left_min_read_len=251
left_max_read_len=251
left_reads_in =396555...
Forum: RNA Sequencing 10-28-2014, 05:48 PM
Replies: 34
Views: 5,489
Posted By Studentlost
Wait I'm sorry, I meant my total reads for each...

Wait I'm sorry, I meant my total reads for each strand are at 1 MB. Each read per file is 251.
Forum: RNA Sequencing 10-28-2014, 05:25 PM
Replies: 34
Views: 5,489
Posted By Studentlost
My reads average around 1,000,000 base pairs. I...

My reads average around 1,000,000 base pairs. I aligned them to the Mmul_1 Rhesus build from Ensembl. I also tried the resMac3 build to compare. I tried with and without a transcriptome index and...
Forum: RNA Sequencing 10-28-2014, 11:11 AM
Replies: 34
Views: 5,489
Posted By Studentlost
There's no reason Tophat should be failing like...

There's no reason Tophat should be failing like this though. Any idea what parameters I can try to change in Tophat to fix the issue?
Forum: RNA Sequencing 10-27-2014, 05:40 PM
Replies: 6
Views: 2,982
Posted By Studentlost
I downloaded the _random chromosomes from the...

I downloaded the _random chromosomes from the UCSC website and built a new Bowtie2 index using this. I ran Tophat with the -G genes.gtf option and the new index.

However....
Here's an error I...
Forum: RNA Sequencing 10-26-2014, 07:23 PM
Replies: 6
Views: 2,982
Posted By Studentlost
Downloaded it again: Here's the chromosome...

Downloaded it again:

Here's the chromosome list:
Mus_musculus/UCSC/mm9/Sequence/Chromosomes/
Mus_musculus/UCSC/mm9/Sequence/Chromosomes/chrY.fa...
Forum: RNA Sequencing 10-26-2014, 06:54 PM
Replies: 6
Views: 2,982
Posted By Studentlost
I am 90% sure that's where I grabbed it from but...

I am 90% sure that's where I grabbed it from but I'll download it again just to verify.
Forum: RNA Sequencing 10-26-2014, 04:04 PM
Replies: 6
Views: 2,982
Posted By Studentlost
Post iGenomes reference genome not accurate?

I grabbed the mm9 reference genome from iGenome. When running cuffcompare, I noticed that the genes.gtf refers to chr#_random whereas the chromosome folder only contained chr#, and no files for...
Forum: RNA Sequencing 10-25-2014, 12:00 PM
Replies: 0
Views: 1,050
Posted By Studentlost
Should jo manager threads match tool threads?

If I assign 12 threads to job manager running my pipeline script, does that mean that tophat and cuffdiff should also have 12 threads assigned to them? Can I use more or less or will it impact...
Forum: RNA Sequencing 10-25-2014, 01:02 AM
Replies: 34
Views: 5,489
Posted By Studentlost
Yeah I did QC before and after checking for...

Yeah I did QC before and after checking for adapter contamination and trimming using scythe and sickle, respectively.

This is really puzzling me. There's no reason for tophat to give me different...
Forum: RNA Sequencing 10-24-2014, 06:41 PM
Replies: 34
Views: 5,489
Posted By Studentlost
The interesting thing is that I took a look at...

The interesting thing is that I took a look at bowtie 2's defaults and tophat was pretty much on point with running them.
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