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Forum: RNA Sequencing 02-12-2015, 04:52 PM
Replies: 2
Views: 2,970
Posted By carmeyeii
I think you misunderstood the OP. He meant having...

I think you misunderstood the OP. He meant having pairs of reads which have become mate-less after QC, not after mapping.

So, one mate will pass QC, but the other one will not, thus leaving you...
Forum: Sample Prep / Library Generation 06-11-2013, 03:47 PM
Replies: 1
Views: 2,122
Posted By carmeyeii
RNAse inhibitors for RNA-seq library prep.

Hi,

I am about to prepare some libraries with precious tissue for RNA-seq. I'd like to protect them as much as I can from RNA degradation, but I'm not positive that all RNAse inhibitors out there...
Forum: Illumina/Solexa 06-05-2013, 01:33 PM
Replies: 58
Views: 39,662
Posted By carmeyeii
Red face :o I know - my bad. i just wanted to say that...

:o
I know - my bad. i just wanted to say that it is the original RNA molecule, without it being "actually the RNA molecule", which of course we all know because this is cDNA we're talking about.;)
Forum: RNA Sequencing 06-05-2013, 01:25 PM
Replies: 4
Views: 1,139
Posted By carmeyeii
:) my brain is now at rest. thank you.

:) my brain is now at rest.

thank you.
Forum: Illumina/Solexa 06-05-2013, 01:09 PM
Replies: 58
Views: 39,662
Posted By carmeyeii
No prob. OK, so just to make sure I'm on the...

No prob.
OK, so just to make sure I'm on the right track -- the sequence that is "spit out" by the sequencer is the actual sequence as seen by the camera... i.e, no base translation, just raw...
Forum: Illumina/Solexa 06-05-2013, 12:54 PM
Replies: 58
Views: 39,662
Posted By carmeyeii
C

kcarr, I thought ScriptSeq's directional protocol is actually tag-based?
I think it doesn't involver dUTP, does it? -- at least ScriptSeq v2 ...
Forum: RNA Sequencing 06-05-2013, 12:50 PM
Replies: 4
Views: 1,139
Posted By carmeyeii
Thanks kmcarr, So the strand with the same...

Thanks kmcarr,

So the strand with the same sequence and direction of the mRNA is the one retained for the first phase of paired-end sequencing, covalently linked through the P7 adapter?

C
Forum: Bioinformatics 06-05-2013, 12:40 PM
Replies: 4
Views: 1,324
Posted By carmeyeii
In DESeq, should one estimate sizeFactors and Dispersion in a pairwise fashion?

When using DESeq, does one also need to import two conditions each time to do the estimatesizefactor and estimatedispersions?

Can one import all conditions (>=3) together, and estimante...
Forum: RNA Sequencing 06-04-2013, 11:49 AM
Replies: 4
Views: 1,139
Posted By carmeyeii
Which strand of the mRNA molecule does the sequencer output as a "read"?

In Illumina Stranded RNA-Seq (using the dUTP method), do the final reads in the fastq files correspond to the initial molecule (that was transcribed), or to the reverse complement of the molecule?
...
Forum: RNA Sequencing 06-04-2013, 11:46 AM
Replies: 0
Views: 1,046
Posted By carmeyeii
What kit would you recommend for Stranded RNA-Seq?

Hi everyone,

I want to prepare several libraries for RNA-seq using the Illumina Hi-Seq.

I am considering several kit options for making said libraries:

Illumina's...
Forum: Illumina/Solexa 06-04-2013, 09:51 AM
Replies: 58
Views: 39,662
Posted By carmeyeii
Is this true? If that is the case, then ...

Is this true?

If that is the case, then

a) the illustration above should portray the complementary strand to the RNA being used as template to synthesize (and read) the actual mRNA molecule by...
Forum: Illumina/Solexa 05-24-2013, 11:01 AM
Replies: 35
Views: 45,306
Posted By carmeyeii
Hi pmiguel Can you expand on this topic a...

Hi pmiguel

Can you expand on this topic a bit?

Thanks,
Carmen
Forum: Illumina/Solexa 05-22-2013, 06:04 PM
Replies: 35
Views: 45,306
Posted By carmeyeii
Illumina has the Stranded and Non-Stranded TruSeq...

Illumina has the Stranded and Non-Stranded TruSeq RNAKits.

i don't know if the adapter Indices are the same for both kits, but I could not find which sequences the Stranded Kits use, until I found...
Forum: Bioinformatics 05-21-2013, 07:04 PM
Replies: 10
Views: 28,127
Posted By carmeyeii
@Dariober, one needs to also append the -t,...

@Dariober, one needs to also append the -t, parameter to your code snippet :) , since it is a comma-separated file and the default delimiter for sort is non-blank to blank.

sort -t, -k1,1...
Forum: Bioinformatics 05-16-2013, 02:22 PM
Replies: 0
Views: 1,090
Posted By carmeyeii
TopHat -g option, --supress-hits, and multiple-mapping reads!?

Hi all,

I am currently using TopHat2 to map RNA-seq runs. I think there have been some changes pertaining the -g option. Does anyone know how it works now?

I used to think that setting -g ...
Forum: Bioinformatics 05-16-2013, 01:19 PM
Replies: 13
Views: 11,964
Posted By carmeyeii
Yes, I think sqcrft is right. It does not discard...

Yes, I think sqcrft is right. It does not discard multiple-mapping reads, but only reports one alignment for those out all all their alignments.

I think there used to be an option -k that would...
Forum: Bioinformatics 05-16-2013, 12:18 PM
Replies: 1
Views: 634
Posted By carmeyeii
My guess is that the information will be...

My guess is that the information will be considered for mapping reads to the GTF file if supplied with -G . In that dataset, read pairs should be concordant with transcript strand. i,e., if...
Forum: Bioinformatics 05-16-2013, 11:59 AM
Replies: 1
Views: 634
Posted By carmeyeii
Does tophat use the library-type information for mapping, or just for the XS flag?

Hi all, quick question:

When I specify library-type to TopHat, i.e., first-strand, second-strand, unstranded, TopHat appends a value + or - to the XS:A tag, which is useful for subsequent...
Forum: Sample Prep / Library Generation 05-15-2013, 06:38 PM
Replies: 35
Views: 17,594
Posted By carmeyeii
Thanks, kcchan! That sums it up :)

Thanks, kcchan!
That sums it up :)
Forum: Illumina/Solexa 05-15-2013, 04:36 PM
Replies: 38
Views: 58,356
Posted By carmeyeii
Also, for anyone looking, the advantages of...

Also, for anyone looking, the advantages of having Y-shaped adapters are explained here:

http://seqanswers.com/forums/showthread.php?t=8869
Forum: Illumina/Solexa 05-15-2013, 04:30 PM
Replies: 38
Views: 58,356
Posted By carmeyeii
Thanks, Phillip, I wonder, though, if that...

Thanks, Phillip,

I wonder, though, if that has changed since the new Y adapters already contain the flowcell-complementary sequence?

Carmen
Forum: Illumina/Solexa 05-15-2013, 04:18 PM
Replies: 12
Views: 50,904
Posted By carmeyeii
HESmith, My thought, actually, is...

HESmith,




My thought, actually, is that upon primer annealing, in each cycle the primer in turn would bind to the start AND end of the DNA fragment, and thus not allow synthesis to continue...
Forum: Bioinformatics 05-15-2013, 12:59 PM
Replies: 3
Views: 1,075
Posted By carmeyeii
i am bumping this up.

i am bumping this up.
Forum: Bioinformatics 05-15-2013, 09:13 AM
Replies: 1
Views: 1,159
Posted By carmeyeii
Hi Sophie, Did you manually enter the...

Hi Sophie,

Did you manually enter the sizeFactors?

If so, I think this is an error that can occur when some sizeFactors are >1.
Forum: Bioinformatics 05-12-2013, 05:47 PM
Replies: 1
Views: 1,643
Posted By carmeyeii
Exclamation Estimating Dispersions DESeq: ALL Conditions at once, or each comparison separately?

Hi everyone,

I have an RNA-seq dataset which comprises 4 different conditions, and 2 biological replicates per condition, like so:

cond1_1 cond1_2 cond2_1 cond2_2 cond3_1...
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