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Forum: RNA Sequencing 09-22-2014, 07:24 PM
Replies: 2
Views: 998
Posted By wacguy
Thank you Gordon, I finally figured it out too. ...

Thank you Gordon, I finally figured it out too.


Guy
Forum: RNA Sequencing 09-15-2014, 01:02 PM
Replies: 2
Views: 998
Posted By wacguy
edgeR contrast

I've noticed that:

lrt <- glmLRT(fit, contrast=c(-1,1,0))

vs

lrt <- glmLRT(fit, contrast=c(1,-1,0))
gives you different set of genes. How do I decide about the order (1, -1 or -1, 1); I...
Forum: Sample Prep / Library Generation 09-05-2014, 06:38 AM
Replies: 268
Views: 100,139
Posted By wacguy
Hi seqgirl, I use a rough estimation from...

Hi seqgirl,

I use a rough estimation from the bioanalyzer (the Qbit minimum RNA amount is too high for such low quantities); if you have single cells RNA, I don't think there is any way to...
Forum: Bioinformatics 08-14-2014, 05:05 AM
Replies: 32
Views: 18,491
Posted By wacguy
-p flag causes problems

Hi,

I'm running tohat2 against the sequence of my transgene (luciferase). I only use the bowtie2 indexes and the FASTA file (i.e., no GTF).
I realized that using the -p 2 (or more) comparing to...
Forum: Sample Prep / Library Generation 06-30-2014, 06:12 PM
Replies: 268
Views: 100,139
Posted By wacguy
Hi konglongjidan, I used the same companies...

Hi konglongjidan,

I used the same companies mentioned in the SMART2-seq article (Biomers & Exiqon). I work at Duke so I guess you would be able to do the same. They were all HPLC purified, I...
Forum: Sample Prep / Library Generation 06-14-2014, 04:52 PM
Replies: 268
Views: 100,139
Posted By wacguy
Thank you Simone!

I appreciate your reply, changing EVERYTHING, and checking the PCR machine was and is my next plan.

Best,
Guy
Forum: Sample Prep / Library Generation 06-14-2014, 07:19 AM
Replies: 268
Views: 100,139
Posted By wacguy
Hi Simone, I've been using your Smart-seq2...

Hi Simone,

I've been using your Smart-seq2 protocol to construct over 20 libraries in the last couple of months w/ great results (Arabidopsois root tissue; around few dozens to few hundreds cells,...
Forum: Sample Prep / Library Generation 02-18-2014, 09:08 AM
Replies: 9
Views: 5,917
Posted By wacguy
Forum: Sample Prep / Library Generation 02-17-2014, 05:04 PM
Replies: 9
Views: 5,917
Posted By wacguy
SMART-seq2 and Nextera XT DNA sample preparation kit

Hi,

I was preparing 3 libraries for RNA-seq starting with 0.03, 0.3 and 3ng total RNA using the modified protocol for SMARTer ultra low (Picelli et al, 2014). I started the...
Forum: Bioinformatics 10-15-2013, 06:18 AM
Replies: 15
Views: 14,763
Posted By wacguy
can you give us a bit more info

which organism, how many samples and replicas, the commands and errors messages. Are you familiar with R?

Guy
Forum: RNA Sequencing 08-27-2013, 04:07 AM
Replies: 3
Views: 1,343
Posted By wacguy
Hi Christa, Yes, I was using it and it all...

Hi Christa,

Yes, I was using it and it all works well (for the Ribo-zero, I use half of the amounts as one of their reps suggested me, and it also saves money). I succefuly ran the Toxedo package...
Forum: RNA Sequencing 08-26-2013, 01:53 PM
Replies: 0
Views: 1,113
Posted By wacguy
Arabidopsis gtf for Cummerbund

Hi,

The Arabidopsis gtf files (NCBI and Ensembl) have some problems (downloaded from iGenome; the NCBI version doesn't give the At numbers for all genes, in the Ensemble version, a gene name "B5...
Forum: Bioinformatics 08-08-2013, 02:02 PM
Replies: 15
Views: 14,763
Posted By wacguy
Hi all, Quite some errors in CummeRbund;...

Hi all,

Quite some errors in CummeRbund; here is the list including the sessionInfo() at the end. I've been trying to solve these isuues for the last 2-3 weeks with little success. Some features...
Forum: RNA Sequencing 07-26-2013, 11:18 AM
Replies: 5
Views: 3,583
Posted By wacguy
did you solve it?

Hi Charitra,

Did you mennage to solve the problem; I am having the same.

Thanks a lot,
Guy
Forum: Bioinformatics 07-22-2013, 01:50 PM
Replies: 0
Views: 492
Posted By wacguy
cuffdiff missing files

Hi,

Count_tracking, read_group_tracking, read_group.info and run.info files are missing after running cuffdiff (4 samples w/ one replica each).

Any idea why?

Thanks a lot,
Guy
Forum: Bioinformatics 07-21-2013, 07:08 PM
Replies: 2
Views: 920
Posted By wacguy
Thanks a lot!!

Thanks a lot!!
Forum: Bioinformatics 07-21-2013, 07:01 AM
Replies: 2
Views: 920
Posted By wacguy
Arabidopsis gtf file with tss and p ids

Hi all,

I'm looking for Arabidopsis gtf file with the tss_is and p_id attributes. all the links I've tried so far seems to lack these fields. I know it is possible to create it with cuffcompare...
Forum: Illumina/Solexa 05-07-2013, 08:33 AM
Replies: 12
Views: 25,385
Posted By wacguy
Hi Shawn, I've seen your reply and wander...

Hi Shawn,

I've seen your reply and wander wether there are new developments in the field such as constructing a directional library rRNA depleted with 1-5ng RNA that are not dependent on pol(A)...
Forum: Sample Prep / Library Generation 03-29-2013, 09:15 AM
Replies: 4
Views: 1,603
Posted By wacguy
Hi Gina, Maybe too late but this week I made...

Hi Gina,

Maybe too late but this week I made a trial library starting the Ribozero with ~10ng! It worked except for some primer dimers, the library was there (Scriptseq V2, Epicentre) as seen by...
Forum: RNA Sequencing 03-23-2013, 10:32 AM
Replies: 3
Views: 1,343
Posted By wacguy
Epicentre kit & Bioanalyzer result

Hi there,

I start constructing RNA-seq libraries after a few month of planning it, from Arabidopsis root material. My initial sample size is very low but using RNAzol, I can get total of 10-15ng...
Forum: RNA Sequencing 12-10-2012, 05:31 AM
Replies: 21
Views: 62,017
Posted By wacguy
RNAzol

Hi Tanals,

I'm using ENAzolRT and it gives really good results; high concentration of RNA (in respect to the small tissue size I am starting with) and good 260/280=1.7 260/230=0.8 values, not...
Forum: RNA Sequencing 10-17-2012, 08:35 AM
Replies: 21
Views: 62,017
Posted By wacguy
Hi Kathie, Thanks a lot for such fast reply....

Hi Kathie,

Thanks a lot for such fast reply. I'm new to RNA world. I used Trizol and no kit to get all RNA types. I ground my small piece of root w/ liquid nitrogen and added the Trizol; (do you...
Forum: RNA Sequencing 10-16-2012, 07:44 PM
Replies: 21
Views: 62,017
Posted By wacguy
Hi Kathie and everyone, Any new insights...

Hi Kathie and everyone,

Any new insights into the low 260/230 ration problem using Trizol? I have identical problem: small sample size> small amount of RNA (which is OK) and very low 260/230 ratio...
Forum: Sample Prep / Library Generation 10-13-2012, 07:03 AM
Replies: 1
Views: 1,272
Posted By wacguy
library prep and...

Hi all,

I'd like to make a library from a small sample size (a few hundred cells) and I'd like to keep strandedness, and to get whole RNA (ie, not using poly(T) but random oligos). After a month...
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