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Forum: Sample Prep / Library Generation 12-14-2017, 05:22 PM
Replies: 5
Views: 2,334
Posted By fbarreto
Hi, sbarberan, We did 14 PCR cycles, with...

Hi, sbarberan,

We did 14 PCR cycles, with total starting RNA of ~700 ng. In consultation with our local sequencing core who does small RNA preps, they suggest these look fine, and we're...
Forum: Sample Prep / Library Generation 12-07-2017, 09:09 AM
Replies: 5
Views: 2,334
Posted By fbarreto
Thank you for your feedback! Looking at the...

Thank you for your feedback! Looking at the TruSeq small RNA guide, it seems this is a common possibility, and may vary with organisms. I will proceed with size-selection.
Forum: Sample Prep / Library Generation 12-06-2017, 10:42 AM
Replies: 5
Views: 2,334
Posted By fbarreto
crustacean small RNA library QC

Hi, everyone,

We're trying for the first time to prepare small RNA libraries for our crustacean model, and we're using the NEBNext small RNA kit. We just finished running the PCR products (before...
Forum: RNA Sequencing 09-10-2013, 06:09 PM
Replies: 0
Views: 2,100
Posted By fbarreto
edgeR: to split or not to split data sets?

Hi all,

I have a specific question regarding matrix design for analyses.
I would like to analyze my samples across two hierarchical levels, and I can think
of at least two ways of performing...
Forum: RNA Sequencing 04-18-2012, 11:17 PM
Replies: 1
Views: 2,706
Posted By fbarreto
Denominator in RPKM calculation

Hi all,

While I understand there are many pros and cons of different normalization methods for RNA-seq gene expression data, I'm currently using RPKM as an initial measure of expression to compare...
Forum: Illumina/Solexa 11-23-2011, 11:52 AM
Replies: 3
Views: 2,278
Posted By fbarreto
I understand how long transcripts show up. I'm...

I understand how long transcripts show up. I'm curious as to how very short transcripts get sequenced through this method (if they do), since some of my genes of interest have short transcripts and...
Forum: Illumina/Solexa 11-23-2011, 11:19 AM
Replies: 3
Views: 2,278
Posted By fbarreto
paired-end RNA-Seq of short transcripts

Hi,

We're planning to start getting ours hands in some of this next-gen stuff, and I have a basic question regarding the paired-end (PE) method for sequencing and mapping in an RNA-Seq experiment....
Forum: Illumina/Solexa 08-03-2011, 06:19 PM
Replies: 4
Views: 18,410
Posted By fbarreto
That's great to know! Thanks a lot! My...

That's great to know! Thanks a lot!
My confusion was that, in my simple mind, the sequencing effort (in bp) versus number of reads had to balance out such that getting longer sequences would mean...
Forum: Illumina/Solexa 08-03-2011, 06:00 PM
Replies: 4
Views: 18,410
Posted By fbarreto
Thanks for insight! But wouldn't a a 2 x 100bp...

Thanks for insight! But wouldn't a a 2 x 100bp paired-end run be sequencing a total of 200bp from each sequence (i.e. transcript copy), 100bp from each end?

So if a run sequences 1,000,000 bp (I...
Forum: Illumina/Solexa 08-03-2011, 04:18 PM
Replies: 4
Views: 18,410
Posted By fbarreto
RNA-seq: read counts in single- vs paired-end sequences

Hi,

I've read several threads with very related topics, but none have addressed the one simple question I have (which stems from my being new to this).

We already have an assembled...
Forum: Bioinformatics 02-26-2010, 01:53 PM
Replies: 4
Views: 2,338
Posted By fbarreto
Thanks! That helps a lot! I tried EMBOSS's...

Thanks! That helps a lot! I tried EMBOSS's needleall, and it does wonderfully in aligning pairs, but I still need to do some scripting to parse that output. Eventually, I want to have each...
Forum: Bioinformatics 02-25-2010, 08:16 PM
Replies: 4
Views: 2,338
Posted By fbarreto
Performing many pairwise alignments

Hi all,

I've stumbled into an issue in my analysis for which I haven't been able to think of a single solution.
I have ~4,000 distinct pairs of orthologous sequences, and I would like to have...
Forum: General 02-20-2010, 05:50 PM
Replies: 2
Views: 2,414
Posted By fbarreto
Re-assembly of singletons?

Hi, all

I have 454 transcriptome data, and I am a bit puzzled by some assemblies I have been doing. When I did a first assembly (on CLC Workbench), the ~600,000 reads assembled into 13,000...
Forum: 454 Pyrosequencing 02-03-2010, 03:34 PM
Replies: 4
Views: 4,810
Posted By fbarreto
Extracting contig info and singletons

Hi,

Our lab recently received 454 sequences back from Roche, and the files came in two types: all reads, and assemblies.
Should we have expected to also receive file(s) containing the singleton...
Forum: Introductions 02-03-2010, 03:11 PM
Replies: 0
Views: 1,232
Posted By fbarreto
New to NextGen

Hello everyone,

I am new postdoc at UC-San Diego, and have for the first started dealing with NextGen sequence data. Learning to use tools for analysing these data has such a steep yet gratifying...
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