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Forum: Sample Prep / Library Generation 08-24-2018, 09:54 AM
Replies: 0
Views: 1,108
Posted By SS00
Hamilton On Deck Thermal Cycler

Does anybody have experience with Hamilton STAR + On Deck Thermal Cycler? How walk-away/automated does this make NGS preps? I'm interested in adding this to our NGS Star used for HT Illumina Preps.
Forum: Bioinformatics 12-15-2016, 02:22 AM
Replies: 0
Views: 1,683
Posted By SS00
Question RNA-seq: Population diversity for a gene based on normalized counts?

Hi all,

A little Bioinformatics/Population Genetics question. I have a list of significant differentially expressed genes from two experimental conditions (each with 5 biological replicates). I...
Forum: Sample Prep / Library Generation 12-30-2015, 10:18 AM
Replies: 7
Views: 1,522
Posted By SS00
Did you do any pre-processing to the RNA like...

Did you do any pre-processing to the RNA like ribo-depletion? I have seen this with my clontech library prep when I used ribo-zero on my RNA before I put it into Clontech's protocol. It seemed that...
Forum: RNA Sequencing 12-08-2014, 03:47 AM
Replies: 1
Views: 1,695
Posted By SS00
Filter cuffcompare by FPKM

How do I filter out low cuffcompare FPKM values out of by combined.gtf file?

I am trying to use the lncrScan workflow for novel long non-coding RNA from total RNA samples. I assembled RABT...
Forum: Bioinformatics 09-09-2014, 01:58 AM
Replies: 6
Views: 2,832
Posted By SS00
Thank you! That makes sense. Yeah, I really...

Thank you! That makes sense.

Yeah, I really have no idea of analysis so I agree my best bet is to work with one of our bioinformaticians and get it sorted.
Forum: Bioinformatics 09-09-2014, 01:39 AM
Replies: 6
Views: 2,832
Posted By SS00
Someone else. I use it on...

Someone else. I use it on https://galaxy.cbio.mskcc.org/ under Differential/Quantitative Analysis.

I used Tophat2 on the same server, downloaded igenomes UCSC genes.gtf and ran DESeq2 and EdgeR...
Forum: Bioinformatics 09-09-2014, 01:33 AM
Replies: 6
Views: 2,832
Posted By SS00
So from the log of EdgeR (but also DESeq2) I can...

So from the log of EdgeR (but also DESeq2) I can see that it gets up to three steps:

% 1. Data preparation %

% 2. Read counting %

% 3. Differential testing %

and then it gives an error at...
Forum: Bioinformatics 09-09-2014, 12:18 AM
Replies: 6
Views: 2,832
Posted By SS00
Angry [bam_parse_region] fail to determine the sequence name

I'm trying to run differential expression using EdgeR and/or DEseq2 on the Ratsch Lab galaxy server but I keep getting this error:



[bam_parse_region] fail to determine the sequence name

...
Forum: Bioinformatics 05-08-2014, 11:00 PM
Replies: 0
Views: 1,257
Posted By SS00
Unhappy TopHat 2 & Tophat for Illumina - Different FPKM?

Hi all,

First let me just say I am new to RNA-seq analysis. I have a weird issue in Galaxy where I am experiencing different transcript expression FPKM values when compared to my reference based...
Forum: Sample Prep / Library Generation 03-24-2014, 05:16 AM
Replies: 6
Views: 8,102
Posted By SS00
Cool So, I just ran two different assays on the same Bioanalyzer Chip.

This was a complete act of desperation but after accidentally running my Agilent RNA Pico Chip on the mRNA assay instead of the Total RNA Assay, and having just used the last ladder aliquot, I left...
Forum: Sample Prep / Library Generation 03-21-2014, 02:59 AM
Replies: 12
Views: 6,347
Posted By SS00
vanillasky, I actually used the Truseq kit but as...

vanillasky, I actually used the Truseq kit but as magnolia93 helpfully explained (thanks!) it is just the fact that you need ligation based adapters for Illumina Sequencing.

magnolia93, when I did...
Forum: Sample Prep / Library Generation 03-20-2014, 01:34 AM
Replies: 12
Views: 6,347
Posted By SS00
I actually had a similar issue and contacted...

I actually had a similar issue and contacted Clontech about it and they didn't really seem surprised and their recommendation was to filter out the adapters. When I brought up the point that I would...
Forum: Sample Prep / Library Generation 03-06-2014, 02:13 AM
Replies: 3
Views: 1,295
Posted By SS00
Ditto for KAPA.

Ditto for KAPA.
Forum: Illumina/Solexa 03-04-2014, 12:44 AM
Replies: 8
Views: 7,358
Posted By SS00
That's interesting. I used the old Truseq Kits,...

That's interesting. I used the old Truseq Kits, including v2, (not old PE kits) without any cycles of PCR using 500ng DNA input and regularly got qPCR values of over 2nM and regular cluster numbers....
Forum: Illumina/Solexa 02-25-2014, 04:23 AM
Replies: 8
Views: 7,358
Posted By SS00
Thumbs up Zaakwalton, has this change from Y-shaped...

Zaakwalton, has this change from Y-shaped adapters been confirmed?

I myself have used the old kit & adapters PCR Free (without one cycle of PCR at the end) with no problems with clustering.
...
Forum: Sample Prep / Library Generation 01-28-2014, 01:25 AM
Replies: 17
Views: 7,191
Posted By SS00
I know this is an old thread but just came across...

I know this is an old thread but just came across this from Agencourt:

https://www.beckmancoulter.com/wsrportal/bibliography?docname=IB-18479.pdf

Seems like you can now select for small RNA's...
Forum: RNA Sequencing 12-11-2013, 12:40 AM
Replies: 9
Views: 3,557
Posted By SS00
I ran a Poly A Prep on Total RNA and found that...

I ran a Poly A Prep on Total RNA and found that even with Poly A selection I still had reads that were mapped as ribosomal. This was really curious, but I guess some rRNA's are polyA?

However,...
Forum: Sample Prep / Library Generation 12-03-2013, 01:04 AM
Replies: 3
Views: 4,124
Posted By SS00
Brill, thanks!

Brill, thanks!
Forum: Sample Prep / Library Generation 12-02-2013, 04:50 AM
Replies: 3
Views: 4,124
Posted By SS00
Can someone explain how Ribo Zero works?

I'm using the Ribo Zero Magnetic Kit from Epicentre and I get that somehow my rRNA ends up bounds to the beads and my ribo-depleted RNA is in the supernatant but I am not sure on the specifics. Can...
Forum: Sample Prep / Library Generation 11-29-2013, 02:06 AM
Replies: 0
Views: 1,609
Posted By SS00
Lightbulb Why can't I just add Poly A to my Total RNA?

Hi all,

I have been weighing up my options with low input RNA. A bit of a debate has been going on regarding Poly A vs Total RNA in my lab. I would love to do Total RNA but I am not in love with...
Forum: Sample Prep / Library Generation 11-22-2013, 12:55 AM
Replies: 1
Views: 1,690
Posted By SS00
Interested in this too. Currently testing...

Interested in this too. Currently testing protocols for Low Input RNA-Seq (500pg-2ng) and have been recommended the TotalScript Nextera by some colleagues.

Up until now I have been testing the...
Forum: Illumina/Solexa 11-06-2013, 06:11 AM
Replies: 2
Views: 3,422
Posted By SS00
Hi Dirk, I have come across this, but...

Hi Dirk,

I have come across this, but didn't find it confusing. I think the reason Illumina asks you to remove the supernatant in two steps is because a 1ml pipette is too big/clumsy to handle...
Forum: Epigenetics 09-23-2013, 09:41 AM
Replies: 0
Views: 2,625
Posted By SS00
Question No DNA in ChIP IgG Control - Strange?

Hi Guys,

Thanks in advance for your help!

Background:
It's my first time doing ChIP. I am testing a native ChIP protocol with MNase digestion using Protein A/G beads and H3K4Me3 and H3K27Me3....
Forum: Sample Prep / Library Generation 09-01-2013, 04:39 AM
Replies: 13
Views: 6,248
Posted By SS00
Thumbs up I have been using the SMARTer kit for RNA prep...

I have been using the SMARTer kit for RNA prep and my lab has a bioruptor. Having recently moved to this lab from a lab with a covaris I will say definitely go for the covaris.

At the moment I am...
Forum: General 07-30-2013, 12:01 AM
Replies: 2
Views: 1,605
Posted By SS00
For high concentrated samples (e.g. with >5cycles...

For high concentrated samples (e.g. with >5cycles PCR) I tend to nanodrop the sample first to get a rough reading of the concentration and dilute down to a range I have confidence that qPCR can...
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