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Forum: Bioinformatics 02-04-2015, 05:56 PM
Replies: 63
Views: 7,540
Posted By mikep
How much QC have you done on the fragmentation...

How much QC have you done on the fragmentation prior to sequencing? It could be the kit, and I say that because a flaky nextera kit on Miseq has turned into a living nightmare for us on a genotyping...
Forum: Bioinformatics 02-04-2015, 04:51 PM
Replies: 9
Views: 4,795
Posted By mikep
Well, you learn something new every day. That...

Well, you learn something new every day. That makes sense. What I meant to say was there is no constant scaling factor between the two, it differs according to the samples.



Fair enuff
Forum: Bioinformatics 02-04-2015, 03:12 PM
Replies: 9
Views: 4,795
Posted By mikep
As I said, normalizing by "total expression" as...

As I said, normalizing by "total expression" as you define it is really just the M part of RPKM. There is no simple way to get TPM from RPKM. You could reverse engineer it if you had the original...
Forum: Bioinformatics 02-03-2015, 09:28 PM
Replies: 9
Views: 4,795
Posted By mikep
TPM (transcripts per million) would be a good way...

TPM (transcripts per million) would be a good way to go, you haven't mentioned how the public data was processed but you could use RSEM to generate the values. I would worry about any between sample...
Forum: Bioinformatics 01-12-2015, 07:59 PM
Replies: 9
Views: 2,248
Posted By mikep
It's been a while since I used IPA, but does it...

It's been a while since I used IPA, but does it have a "this data came from RNAseq" check button when you upload your DEGlist? I'm guessing not, but even if it did, the "length of a gene" is...
Forum: Bioinformatics 01-11-2015, 05:19 PM
Replies: 1
Views: 1,170
Posted By mikep
Meme will look for enriched motifs in your...

Meme will look for enriched motifs in your sequence, no guarantees said motifs mean anything, or represent any particular TF. transfac will look for known TF sites in your data and report them,...
Forum: Bioinformatics 12-11-2014, 09:22 PM
Replies: 8
Views: 11,119
Posted By mikep
If you have a small initial sample you want it as...

If you have a small initial sample you want it as enriched as possible for the stuff you actually care about. For most purposes, the non mRNA you get from totalRNA is a nice to have, not a must...
Forum: Bioinformatics 12-11-2014, 08:43 PM
Replies: 8
Views: 11,119
Posted By mikep
It can also depend on your sample. If you have...

It can also depend on your sample. If you have miniscule starting material and need some serious amplification then the protocol may require mRNA as a starting point. In addition if you are going...
Forum: Bioinformatics 10-16-2014, 02:51 PM
Replies: 4
Views: 1,548
Posted By mikep
Well here's the page on htseq-count ...

Well here's the page on htseq-count

http://www-huber.embl.de/users/anders/HTSeq/doc/count.html

With pretty simple instructions. The output is raw counts per gene, which you can then feed...
Forum: Bioinformatics 10-16-2014, 02:01 PM
Replies: 4
Views: 1,548
Posted By mikep
Yes you can. You just need a script to determine...

Yes you can. You just need a script to determine counts per gene (like htseq-count), then you are good to go.
Forum: Bioinformatics 10-01-2014, 12:36 AM
Replies: 3
Views: 1,669
Posted By mikep
The SRA is arcane. biosample refers to the...

The SRA is arcane.

biosample refers to the source of your material. If it was 8 different people then that is 8 different biosamples. if it was, say, 2 people which were given a time course of...
Forum: Bioinformatics 08-18-2014, 01:22 AM
Replies: 18
Views: 8,263
Posted By mikep
That is unfortunate. You are however correct,...

That is unfortunate. You are however correct, further validation is absolutely required.

I have a question, how many genes pass uncorrected pv 0.05 and what is the range of the fold change you...
Forum: Bioinformatics 08-07-2014, 07:19 PM
Replies: 18
Views: 8,263
Posted By mikep
Well I'm a fan of parametric over parametric,...

Well I'm a fan of parametric over parametric, what I was suggesting is a better design in which you take the paired nature of the data into account

... my R is crap but something like design <-...
Forum: Bioinformatics 08-07-2014, 07:16 PM
Replies: 9
Views: 2,269
Posted By mikep
I normally get about a 10% miss rate with...

I normally get about a 10% miss rate with mapping, finished a bunch of star runs this morning to find a miss rate of 25%.

If I find anything in it I'll get back, otherwise 'fraid I got nothing.
Forum: Bioinformatics 08-07-2014, 01:16 AM
Replies: 9
Views: 2,269
Posted By mikep
Well, I dunno what bowtie2 is doing, but that...

Well, I dunno what bowtie2 is doing, but that first sequence you posted above has a 100% hit to various bacterial sequences, and no hits to human using megablast, so I'd be rather glad star aint...
Forum: Bioinformatics 08-07-2014, 12:11 AM
Replies: 9
Views: 2,269
Posted By mikep
Did you mean you look at the leftover reads (as...

Did you mean you look at the leftover reads (as opposed to transcripts)?

Also, whats the quality like on those reads, and what do the bowtie alignments look like?
Forum: Bioinformatics 08-07-2014, 12:04 AM
Replies: 1
Views: 1,394
Posted By mikep
Did you try cufflinks with -no-novel-juncs, and...

Did you try cufflinks with -no-novel-juncs, and tell it to look just for known transcripts? is alternate splicing something you actually need to worry about? Maybe htseq-count and edger/deseq2 is a...
Forum: Bioinformatics 08-06-2014, 11:19 PM
Replies: 18
Views: 8,263
Posted By mikep
Actually, what you describe are replicates. It...

Actually, what you describe are replicates. It just so happens that the biological variability is overwhelming your treatment variability.

Did you try a paired analysis?

As mentioned above, if...
Forum: Bioinformatics 08-06-2014, 12:59 AM
Replies: 7
Views: 5,182
Posted By mikep
You didn't mention your sample source. If it is...

You didn't mention your sample source. If it is different people then 0.93 might be as good as it gets. I get around 0.95 on my data.

Another option (for future use) is to use a spikein like...
Forum: Bioinformatics 08-05-2014, 06:03 PM
Replies: 7
Views: 5,182
Posted By mikep
Raw counts don't follow a linear distribution. ...

Raw counts don't follow a linear distribution. Use Spearman, not Pearson. And discard any genes with 0 counts. Actually, I'd probably discard genes with < 10.

Secondly, is this human data, or...
Forum: Bioinformatics 08-04-2014, 07:03 PM
Replies: 1
Views: 2,362
Posted By mikep
1) Change the phase from "0" to "." (may not...

1) Change the phase from "0" to "." (may not make a difference, but it's what I use)
2) Do you have features in the gtf of type "transcript" (as opposed to exon)?
Forum: Bioinformatics 08-04-2014, 06:55 PM
Replies: 4
Views: 1,374
Posted By mikep
There are some differential engines which can...

There are some differential engines which can distinguish between technical and biological replicates, or you could merge them. However, the larger issue is "the first one yielded very few reads"
...
Forum: Bioinformatics 07-06-2014, 09:16 PM
Replies: 3
Views: 1,341
Posted By mikep
Compile all the genomes that are present into a...

Compile all the genomes that are present into a fasta file, build a bowtie index against it and then run bowtie (or do a similar thing for which ever mapper you prefer).

We've has success doing...
Forum: Bioinformatics 06-02-2014, 11:58 PM
Replies: 6
Views: 2,025
Posted By mikep
Ummm.... you are in trouble. I'd dump all...

Ummm.... you are in trouble.

I'd dump all the reads from all experiments into 1 big file then do a de novo assembly.

Then turn that into your reference genome, and map each sample back...
Forum: Bioinformatics 06-02-2014, 11:20 PM
Replies: 9
Views: 5,755
Posted By mikep
Alot of insects have Wolbachia integrated into...

Alot of insects have Wolbachia integrated into their chromosomes. Might not be contamination/infection.
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