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Forum: Bioinformatics 07-30-2012, 05:33 PM
Replies: 2
Views: 2,343
Posted By sjm
Maybe take a look at this paper: ...

Maybe take a look at this paper:

http://www.ncbi.nlm.nih.gov/pubmed/21171994

This will get you 'count' data for your microRNAs - I then use DESeq to perform differential gene expression.
Forum: RNA Sequencing 06-24-2012, 02:04 PM
Replies: 3
Views: 3,786
Posted By sjm
I like the potassium and magnesium acetate salts...

I like the potassium and magnesium acetate salts + heat method in Mortazavi et al., Nat Methods 2008. Zymo RNA Clean and Concentrator columns make a nice alternative to purifying your RNA afterward...
Forum: RNA Sequencing 04-11-2012, 05:21 AM
Replies: 10
Views: 7,532
Posted By sjm
These are great points on the MIQE guidelines and...

These are great points on the MIQE guidelines and the necessity of doing qPCR the right way or not at all. Dvanic, I share your pain about the need for multiple reference genes (I've had a very hard...
Forum: RNA Sequencing 04-02-2012, 03:46 PM
Replies: 1
Views: 1,637
Posted By sjm
I think as a first step, you will indeed need to...

I think as a first step, you will indeed need to have bowtie build a database from your fasta file.

bowtie-build /path/to/fasta name_of_database

Then have tophat align your reads to that...
Forum: RNA Sequencing 04-01-2012, 06:01 AM
Replies: 1
Views: 1,591
Posted By sjm
Your concentrations seem to be quite high - I use...

Your concentrations seem to be quite high - I use the same kit, and have been achieving 10-20 ng/uL (in a 10 uL) volume from mouse and rat pancreatic tissue. I don't have my lab notebook in front of...
Forum: RNA Sequencing 03-31-2012, 08:42 AM
Replies: 0
Views: 2,641
Posted By sjm
many biological replicates - 'traditional' statistics vs Cuffdiff or DESeq/edgeR?

I've read the discussions from Simon Anders and Lior Pachter intensively on these forums and understood them as best I can without extensive graduate training in mathematics or statistics :-) I...
Forum: RNA Sequencing 03-31-2012, 08:12 AM
Replies: 0
Views: 2,591
Posted By sjm
multiple pairwise comparisons in DESeq and/or edgeR

Hi all,

I have a study involving multiple treatment groups (A, B, C and D) vs a baseline condition (Z). There are 6 biological replicates in each treatment group. I'm interested in using DESeq...
Forum: RNA Sequencing 03-31-2012, 07:58 AM
Replies: 10
Views: 7,532
Posted By sjm
validation of RNASeq results

My two cents' worth is that RNA-seq is in general a vastly superior technique to RT-qPCR, and having published a few papers now involving RNA-seq methods, I"m becoming frustrated with reviewers...
Forum: RNA Sequencing 03-31-2012, 07:45 AM
Replies: 1
Views: 3,230
Posted By sjm
E-miR program

Try taking a look at the article below. I've been using this pipeline to analyze miR sequencing reads generated via Illumina's TruSeq small RNA preparation kit, on an Illumina HiSeq. The software...
Forum: Bioinformatics 12-01-2011, 06:09 AM
Replies: 2
Views: 1,063
Posted By sjm
tophat segment error -9 due to memory problems?

@crader,

I had a number of similar problems when running tophat on a desktop machine with only 4 G of memory, and a misconfigured swap file (only 2 G in size). I don't know how big your input...
Forum: RNA Sequencing 05-27-2011, 07:29 PM
Replies: 13
Views: 11,315
Posted By sjm
paired-end data in correct format?

@edge: are you sure that your 2x50 bp paired-end data are in the correct format for Cufflinks, i.e. are you choosing the right --library-type option? Sorry I can't be more helpful, as I haven't...
Forum: RNA Sequencing 05-27-2011, 01:33 PM
Replies: 13
Views: 11,315
Posted By sjm
Tophat bam and sam files

I could be wrong, but my understanding is that the current version of tophat only produces files in .bam format. However, it's easy enough to convert back later - you already have the samtools...
Forum: RNA Sequencing 06-08-2010, 04:34 PM
Replies: 2
Views: 4,647
Posted By sjm
advice on tophat/cufflinks genes/transcripts and RPKM

I've seen similar behavior when using cufflinks to look at RPKM assigned to Ensembl transcript entries. If you look carefully at exactly which chromosomal locations are involved for each transcript...
Forum: RNA Sequencing 06-08-2010, 04:16 PM
Replies: 2
Views: 2,967
Posted By sjm
I think I had similar issues with TopHat when I...

I think I had similar issues with TopHat when I was first using it a year or so ago. Check the chromosome descriptors in your gff3 file and make sure they match those in your bowtie index (you can...
Forum: RNA Sequencing 06-08-2010, 04:01 PM
Replies: 11
Views: 5,672
Posted By sjm
analysis of biological replicates (groups) via Tophat/Cufflinks

Hi,

Sorry that I haven't posted for a while. blackgore, for analysis of replicates, I did not use Tophat/Cufflinks for this part of the operation. Having produced a list of genes/transcripts and...
Forum: RNA Sequencing 01-20-2010, 05:09 AM
Replies: 2
Views: 4,293
Posted By sjm
Thanks for the advice - I'll check recent...

Thanks for the advice - I'll check recent Bioinformatics issues.

I would still be interested to hear from anyone who has insight into how Tophat/Cufflinks (specifically) deals with this problem......
Forum: Bioinformatics 01-14-2010, 04:45 AM
Replies: 34
Views: 5,875
Posted By sjm
Yes, I can confirm that your .3.ebwt file is OK. ...

Yes, I can confirm that your .3.ebwt file is OK. I have a bunch of bowtie indexes for mouse (self-built from Ensembl databases) and the .3 file is always a few kb only.
Forum: Bioinformatics 01-13-2010, 05:44 PM
Replies: 34
Views: 5,875
Posted By sjm
Hmm, I've never tried tophat with such large...

Hmm, I've never tried tophat with such large fastq files. The largest I've tried has been 1.5G. Maybe you should get in touch with Cole Trapnell, the guy who largely wrote Tophat, and see if...
Forum: Bioinformatics 01-13-2010, 03:45 PM
Replies: 34
Views: 5,875
Posted By sjm
By the way, there are newer versions of both...

By the way, there are newer versions of both bowtie and tophat available for download and the authors have squashed a few bugs. Probably not relevant to your error, but worth having the latest.
Forum: Bioinformatics 01-13-2010, 03:44 PM
Replies: 34
Views: 5,875
Posted By sjm
Do you have bowtie in your PATH?

Do you have bowtie in your PATH?
Forum: Bioinformatics 01-13-2010, 03:42 PM
Replies: 1
Views: 1,858
Posted By sjm
I don't think there's anything you need to...

I don't think there's anything you need to specify. I don't have paired-end reads and have been running Cufflinks to help with RNASeq analysis for a few months now. Just try the default options...
Forum: RNA Sequencing 01-13-2010, 03:38 PM
Replies: 13
Views: 11,315
Posted By sjm
have you thought of trying the cufflinks module?

Getting GFF3-formatted files, or constructing them from gtfs, is a real pain. Been there, done that, for the mouse genome. Have you considered using Cufflinks, from the authors that produced...
Forum: RNA Sequencing 12-18-2009, 01:12 PM
Replies: 11
Views: 5,672
Posted By sjm
Great - let's work on getting you some data to...

Great - let's work on getting you some data to play with. These are data that I am working up for publication, so if it doesn't mess up your calculations and you're OK with my data being...
Forum: RNA Sequencing 12-17-2009, 08:23 AM
Replies: 11
Views: 5,672
Posted By sjm
By the way, you may also be interested to know...

By the way, you may also be interested to know that we've multiplexed 4 samples per Illumina GAII lane (i.e. barcoding system), but haven't tried examining single samples per lane.
Forum: RNA Sequencing 12-17-2009, 08:22 AM
Replies: 11
Views: 5,672
Posted By sjm
Well, maybe everyone isn't using biological...

Well, maybe everyone isn't using biological replicates, but I certainly am in experiments involving RNASeq of nontransgenic and transgenic mouse tissues...!
I could send you RPKM expression values...
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