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Forum: Bioinformatics 04-22-2018, 11:26 PM
Replies: 0
Views: 542
Posted By buthercup_ch
correlation among biological replicates

Hello,

I am trying to analyze some data that I just got from RNA-Seq from gram-negative bacteria.

The bioinformatician just gave me 2 sets of tables 1) reads counts, 2) FPKM.
Sequences has...
Forum: Bioinformatics 10-03-2016, 07:58 AM
Replies: 3
Views: 1,071
Posted By buthercup_ch
Time Course RNA-Seq

Hello,

We just finished the QC for the total reads obtain from a Time Course RNA-Seq experiment (Illumina HiSeq2000), and are satisfied with the quality.
Now we have started the Differential...
Forum: RNA Sequencing 09-29-2016, 01:51 AM
Replies: 7
Views: 1,496
Posted By buthercup_ch
Hello GenoMax, The procedures in the Core...

Hello GenoMax,

The procedures in the Core Facility are in fact slightly dark for me.
Nevertheless, thanks for your comments and advised. At first, analysis was carried out trimming the first 3 nt...
Forum: RNA Sequencing 09-28-2016, 06:49 AM
Replies: 7
Views: 1,496
Posted By buthercup_ch
Hello, Thanks for your comments. I...

Hello,

Thanks for your comments.

I agree trimming will improve the overall quality of the reads set.
But what about the Quality per tile diagram? It is the first time I face such a pattern,...
Forum: RNA Sequencing 09-27-2016, 02:22 PM
Replies: 7
Views: 1,496
Posted By buthercup_ch
FASTQC reports

Hello everyone,

I just received a set of sequences from an RNA-Seq experiment.
Illumina HiSeq 2000, pair-end sequencing was performed.

Quality of R1 sequences look good, but R2 not so......
Forum: RNA Sequencing 09-27-2016, 01:10 PM
Replies: 4
Views: 1,342
Posted By buthercup_ch
Hello fanli, Thanks for your message. To...

Hello fanli,

Thanks for your message.
To answer your question, yes the Ribozero kit used was appropriate for our samples. It was the Ribo-Zero Gold rRNA Removal Kit (Epidemiology). This kit...
Forum: RNA Sequencing 09-26-2016, 08:37 AM
Replies: 4
Views: 1,342
Posted By buthercup_ch
High number of read counts mapping to bacterial rRNA genes in RNA-Seq experiment

Hello to everyone.

In my group, we are using RNA-Seq to study the expression of bacteria under different conditions, isolated or as dual-transcriptome when in association with plants.

When...
Forum: Illumina/Solexa 08-09-2016, 11:35 PM
Replies: 2
Views: 2,297
Posted By buthercup_ch
Illumina V3 vs. V4 chemistry

Hello everyone.

We are dealing with RNA-Seq of bacteria.
So far, we were using HiSeq2000/2500... with V3 chemistry. But recently the genomic platform has the HiSeq4000 available... which runs...
Forum: Sample Prep / Library Generation 08-01-2016, 12:38 AM
Replies: 1
Views: 1,360
Posted By buthercup_ch
RiboZero kits

Hello,

After looking on the web, I haven't been really successful in finding information about the difference among the several rRNA removal kits from Epicentre (I think it was recently absorbed...
Forum: Sample Prep / Library Generation 07-28-2016, 06:50 AM
Replies: 5
Views: 2,335
Posted By buthercup_ch
Exactly, that is my main concern... As I don't...

Exactly, that is my main concern... As I don't have that much experience and never yet faced this scenario..., I am not sure if the libraries will be good for sequencing.

They look pretty good as...
Forum: Sample Prep / Library Generation 07-28-2016, 06:16 AM
Replies: 10
Views: 3,147
Posted By buthercup_ch
Thanks nucacidhunter for the clarification. ...

Thanks nucacidhunter for the clarification.

We started with 500 ng... so I guess a low input was not the case of "failure"... I did not perform the preparation, but I guess a combination of low...
Forum: Sample Prep / Library Generation 07-28-2016, 05:07 AM
Replies: 10
Views: 3,147
Posted By buthercup_ch
hello nucacidhunter, thanks for your comment. ...

hello nucacidhunter,
thanks for your comment.

Please, I am getting a bit lost with the vocabulary... what do you mean by "low input" and 'high input"?

Total RNA samples were of high quality...
Forum: Sample Prep / Library Generation 07-28-2016, 04:55 AM
Replies: 10
Views: 3,147
Posted By buthercup_ch
Thank you so much jdk787 for your clarifications....

Thank you so much jdk787 for your clarifications.

We will try to clean-up the libraries once more. I hope we'll not loose too much material in the process.
Forum: Sample Prep / Library Generation 07-28-2016, 04:43 AM
Replies: 5
Views: 2,335
Posted By buthercup_ch
Hello jteeee2, We are working with...

Hello jteeee2,

We are working with prokaryotes, and the protocol followed for library preparation was:
- Ribo-Zero Magnetic Gold
- TruSeq Stranded mRNA Sample Preparation

In a previous...
Forum: Sample Prep / Library Generation 07-27-2016, 09:00 AM
Replies: 5
Views: 2,335
Posted By buthercup_ch
RNA-Seq library size

Hello,

Does any have strong experience and can give good advice regarding the optimal concentration of libraries for RNA-Seq?

I normally have worked with 30-50 ng/ul, but in the last set of...
Forum: Sample Prep / Library Generation 07-27-2016, 08:54 AM
Replies: 2
Views: 1,267
Posted By buthercup_ch
Sorry but this amples are supposed to be total...

Sorry but this amples are supposed to be total RNA, right?
Then why observing 2 peaks is strange? you should observe several peaks actually, mainly those for 16S and 23S if working with bacteria (as...
Forum: Sample Prep / Library Generation 07-27-2016, 07:37 AM
Replies: 10
Views: 3,147
Posted By buthercup_ch
Hi jdk787, Thanks for your answer. Do you...

Hi jdk787,

Thanks for your answer.
Do you mean to use AMPure XP beads once more?
Just to clarify, 0.8X means the ratio of volume to be mixed right? 0.8 vol beads + 1 vol library?

I am also...
Forum: Sample Prep / Library Generation 07-27-2016, 07:12 AM
Replies: 2
Views: 806
Posted By buthercup_ch
What is the meaning of "keep the OD of the...

What is the meaning of "keep the OD of the culture the same throughout"?

You are going to study the differential expression between control and treated samples, right? Then of course bacteria will...
Forum: Sample Prep / Library Generation 07-27-2016, 06:28 AM
Replies: 10
Views: 3,147
Posted By buthercup_ch
Primer dimer contamination

Hello,

I would like to ask for the opinion of those having experience in this procedure.
We are dealing with bacterial transcriptomics and applying RNA-Seq.

We are following the TruSeq RNA...
Forum: Illumina/Solexa 07-26-2016, 10:29 PM
Replies: 4
Views: 1,693
Posted By buthercup_ch
Thanks GenoMax for your comments. The actual...

Thanks GenoMax for your comments.

The actual size of the insert is 300 bp, what we have been using with 2000/2500.
Please can you explain in more details why the insert size is constraint in such...
Forum: Illumina/Solexa 07-26-2016, 01:12 AM
Replies: 4
Views: 1,693
Posted By buthercup_ch
HiSeq 4000 upgrade

Hello,

We have been running RNA-Seq in HiSeq2000/2500 and are quite happy with the results.
But there is now just available the HiSeq4000.

Please, any one having experience with the new...
Forum: RNA Sequencing 06-19-2016, 07:42 AM
Replies: 1
Views: 1,422
Posted By buthercup_ch
Input BAM files for Cufflinks

Hello,

Can I use the BAM file obtained after mapping of RNA-Seq reads using CLC Genomics Workbench as input for Cufflinks, in order to analyze differential expression?

Thanks
Forum: Bioinformatics 06-18-2016, 01:44 PM
Replies: 0
Views: 1,154
Posted By buthercup_ch
EDGE-pro: Estimated Degree of Gene Expression in Prokaryotic Genomes

Hello,

We are studying the differential gene expression of bacteria under different growth conditions, by applying RNA-Seq.
The pipeline she used so far is the following:
- After quality check...
Forum: Bioinformatics 10-31-2014, 03:10 AM
Replies: 1
Views: 872
Posted By buthercup_ch
Identification and grouping of ortholog protein from a set of prokaryotic genomes

Hi everyone.

In the frame of a project of comparative genomics, I have right now a set of about 30 whole-genome sequences of bacteria, of about 3-4 Gb size, and need to retrieve orthologous groups...
Forum: Bioinformatics 10-21-2014, 12:26 AM
Replies: 1
Views: 793
Posted By buthercup_ch
Normalization of RNA-seq bias

Hi everyone.
I'm have a question…, very basic I'm afraid.
I just received the results of my RNA-seq sequencing reaction, mate-pair, and it seems that there was a problem during read 2 sequencing....
Showing results 1 to 25 of 40

 


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