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Forum: RNA Sequencing 10-22-2013, 02:55 PM
Replies: 6
Views: 1,596
Posted By tboothby
No, not yet. I am planning on trying to confirm...

No, not yet. I am planning on trying to confirm some of my RNAseq data with qPCR.



Desiccation stress... the organism dried out and becomes ametabolic, after rehydration it recommences...
Forum: Bioinformatics 10-22-2013, 02:49 PM
Replies: 0
Views: 995
Posted By tboothby
SNP or paralog?

Hi all,
I have a genome in a state of partial assembly and a transcriptome that has been de novo assembled.

I am curious if I can tell the difference between an allelic SNP and a recent gene...
Forum: RNA Sequencing 10-21-2013, 06:07 AM
Replies: 6
Views: 1,596
Posted By tboothby
Thanks for the reply. Yes I have been looking at...

Thanks for the reply. Yes I have been looking at the raw reads and they tell the same story. In my stressed condition the raw reads are very low for things I would expect to be high, relative to...
Forum: RNA Sequencing 10-21-2013, 04:38 AM
Replies: 6
Views: 1,596
Posted By tboothby
Unexpected differential expression

I was wondering if anyone had any input on what follows below:

I have a pretty straightforward experimental setup for de novo transcriptome assembly and differential expression analysis:

2...
Forum: Bioinformatics 09-06-2013, 09:16 AM
Replies: 1
Views: 1,769
Posted By tboothby
Redundancy vs. Artifacts in de novo transcriptomes

Hey all,

I was wondering if anyone knows of (or can think of) a way to assess whether isoforms in a de novo transcriptome assembly are real or an artifact of sequencing/assembly.

I was thinking...
Forum: Bioinformatics 09-03-2013, 01:37 PM
Replies: 2
Views: 1,932
Posted By tboothby
Selecting sequencing reads based on kmer counts

Forgive my ignorance but is there a established method for selecting sequencing reads based on kmer count?

e.g., I want to parse only sequencing reads that contain kmer sequences with between...
Forum: Bioinformatics 08-06-2013, 07:27 AM
Replies: 36
Views: 8,756
Posted By tboothby
Would somebody who has successfully built HTSeq...

Would somebody who has successfully built HTSeq on a Mac mind sharing their build with me? I am getting the same error as in the original post, unfortunately the subsequent suggestions don't seem to...
Forum: Bioinformatics 07-29-2013, 09:23 AM
Replies: 3
Views: 947
Posted By tboothby
Find GO terms sharing transcripts

Hi,
Does anybody know if a tool exists for looking that the distribution of transcripts to GO terms?

Basically I have BLASTed a bunch of transcripts from and RNAseq project (non-model organism)...
Forum: Bioinformatics 02-21-2012, 06:39 AM
Replies: 5
Views: 1,436
Posted By tboothby
I am using the latest release of BLAST+ ...

I am using the latest release of BLAST+

Command line:
blastx -db databases/refseq_protein -gilist GI_hyp_put_pre_unchar -query...
Forum: Bioinformatics 02-20-2012, 12:31 PM
Replies: 5
Views: 1,436
Posted By tboothby
Weird Tabular BLAST Occurrence

Something odd I just found while performing a stand alone BLAST. I have the number of descriptions set to 10, which is the number of hits reported for most of my queries... but one of my queries has...
Forum: Bioinformatics 02-03-2012, 10:06 AM
Replies: 1
Views: 1,893
Posted By tboothby
Stand Alone Blast Exclude without -remote

Does anyone know if there is a way to exclude certain search perimeters from a Stand Alone Blast?

I know you can do with using the Entrez Query options (either in the web or Stand Alone Version)....
Forum: RNA Sequencing 02-03-2012, 09:24 AM
Replies: 25
Views: 6,480
Posted By tboothby
Unfortunately, we don't.

Unfortunately, we don't.
Forum: RNA Sequencing 02-03-2012, 08:32 AM
Replies: 25
Views: 6,480
Posted By tboothby
Our "reference transcriptome" was assembled using...

Our "reference transcriptome" was assembled using reads combined across all time points of development. We wanted to be able to map reads from specific time points to the reference, along with...
Forum: RNA Sequencing 02-03-2012, 07:50 AM
Replies: 25
Views: 6,480
Posted By tboothby
Using this approach have you seen any issues with...

Using this approach have you seen any issues with cufflinks assembly of transcripts from your mapped reads? We tried the same approach and found an inverse relationship between mapping coverage and...
Forum: Bioinformatics 02-01-2012, 08:15 AM
Replies: 16
Views: 7,003
Posted By tboothby
My initial thought is that abundant transcripts...

My initial thought is that abundant transcripts are generating a lot of sequence reads (obviously) and that the de novo assembler is making many (potentially erroneous) isoforms for those...
Forum: Bioinformatics 01-26-2012, 04:45 AM
Replies: 2
Views: 7,088
Posted By tboothby
Another few questions: I have used Bowtie to...

Another few questions:
I have used Bowtie to map read fragments to a reference transcriptome. When I look at a sample of individual transcripts in IGV I see they have thousand fold coverage.

When...
Forum: Bioinformatics 01-25-2012, 10:16 AM
Replies: 2
Views: 7,088
Posted By tboothby
RSEM expected counts question

I want to check that I understand the output of RSEM correctly. As I understand it the "expected_count" output for each gene is the number of fragment reads that are predicted to map to that "gene." ...
Forum: Bioinformatics 01-24-2012, 12:44 PM
Replies: 16
Views: 7,003
Posted By tboothby
Mike, Have you tried using cufflinks to...

Mike,
Have you tried using cufflinks to assemble transcripts with your mapped reads yet?

If so, do you see any instances where transcripts from your de novo transcriptome have good mapping...
Forum: Bioinformatics 01-23-2012, 01:04 PM
Replies: 8
Views: 5,000
Posted By tboothby
Are you familiar with unix? Try a google search...

Are you familiar with unix? Try a google search for the awk command. Very powerful tool.
Forum: Bioinformatics 01-23-2012, 12:02 PM
Replies: 16
Views: 7,003
Posted By tboothby
@Mike As Westerman points out the Trinity...

@Mike
As Westerman points out the Trinity website now has some basic procedures for aligning read fragments to a Trinity transcriptome.

They suggest using bowtie to align read fragments and then...
Forum: Bioinformatics 01-23-2012, 08:20 AM
Replies: 16
Views: 7,003
Posted By tboothby
Hi Mike, I am currently trying to do the same...

Hi Mike,
I am currently trying to do the same thing (use a de novo transcriptome as a reference for Tophat and cufflinks). I am not sure about your 'N' problem.

With your header issue have you...
Forum: Bioinformatics 01-20-2012, 08:35 AM
Replies: 1
Views: 1,290
Posted By tboothby
Tophat -> Cuffdiff missing sequences

Hi all. I am hoping you guys can help me with an issue, I apologize for my newbish-ness ahead of time, I am still trying to learn the software.

Background:
I run tophat/bowtie on a RNAseq sample...
Forum: Bioinformatics 01-18-2012, 10:20 AM
Replies: 20
Views: 24,090
Posted By tboothby
Hi, I am using blast+. I have formatted a...

Hi,
I am using blast+. I have formatted a nucleotide database using makeblastdb.

I am trying to extract sequences from a file containing a list of IDs.

Using the...
Forum: Bioinformatics 01-16-2012, 07:52 AM
Replies: 23
Views: 42,752
Posted By tboothby
I am trying the following command and...

I am trying the following command



and I get the following:


Edit: I find it strange that contig names are different... I am going rerun tophat and see if the this difference in naming...
Forum: Bioinformatics 01-16-2012, 06:55 AM
Replies: 23
Views: 42,752
Posted By tboothby
OK, I have two conditions I want to test. I ran...

OK, I have two conditions I want to test. I ran them through tophat and then did the following for each:



I thought this would sort my two files into the same order. Below are the first few...
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