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Forum: Illumina/Solexa 02-25-2020, 10:58 PM
Replies: 3
Views: 1,278
Posted By GSviral
Hello mdegraaf, You are correct with your...

Hello mdegraaf,

You are correct with your assumption. The PE 8 + 8 option refers to a dual indexing strategy while the PE 8 option will just be a single indexing approach. However, if you are...
Forum: Bioinformatics 02-19-2020, 10:51 PM
Replies: 3
Views: 860
Posted By GSviral
For mapping and alignment I would consider them...

For mapping and alignment I would consider them interchangeable with regards to a reference genome, yeah. If I mentioned either to my colleagues they would understand both just the same. Just the...
Forum: Bioinformatics 02-19-2020, 03:31 AM
Replies: 3
Views: 860
Posted By GSviral
For me, I would interpret read mapping as...

For me, I would interpret read mapping as aligning (or mapping) the reads against a reference genome. Putting the reads together to form contigs would be an assembly.

Contig alignment, to me,...
Forum: Illumina/Solexa 10-15-2019, 10:59 PM
Replies: 2
Views: 1,452
Posted By GSviral
Hi kgoglin, When I previously used the KAPA...

Hi kgoglin,

When I previously used the KAPA qPCR quantification method I also ran into the same problem from time to time. Sometimes triplicate standards could be very variable between runs even...
Forum: Sample Prep / Library Generation 04-09-2019, 12:05 AM
Replies: 4
Views: 1,476
Posted By GSviral
Hi there, Off the top of my head I think I...

Hi there,

Off the top of my head I think I am using a bottle, stored at +4, that is currently sitting older than 18 months and they have performed perfectly in all my recent preps so I'd say...
Forum: Illumina/Solexa 02-21-2019, 11:06 PM
Replies: 13
Views: 10,235
Posted By GSviral
Hi kternus, I use the Nextera XT library...

Hi kternus,

I use the Nextera XT library prep kit with 15 cycles of PCR. Originally, it was for low concentration viral DNA/cDNA and I found it helped boost the yield after the index PCR to get...
Forum: Sample Prep / Library Generation 11-19-2018, 12:19 AM
Replies: 4
Views: 1,552
Posted By GSviral
Hi GenomeM, Thanks for giving an update on...

Hi GenomeM,

Thanks for giving an update on this, it's good to know that the RSB didn't affect the binding! I'll keep that in mind for the future.

Cheers.
Forum: Illumina/Solexa 11-13-2018, 11:32 PM
Replies: 3
Views: 935
Posted By GSviral
Good to hear that those higher cluster densities...

Good to hear that those higher cluster densities can still produce good quality data.

Unfortunately it's hard to predict, one library can produce completely different metrics to another at those...
Forum: Illumina/Solexa 11-13-2018, 11:27 PM
Replies: 4
Views: 1,571
Posted By GSviral
Hi Gaetan, Yeah it's always a bother when...

Hi Gaetan,

Yeah it's always a bother when working with precious samples and NGS! I took a quick look and found this from August 2018:
...
Forum: Sample Prep / Library Generation 11-13-2018, 06:26 AM
Replies: 4
Views: 1,552
Posted By GSviral
Hi, I'm not too sure about the resuspension...

Hi,

I'm not too sure about the resuspension buffer preventing the binding to the RNA beads, but in any TruSeq protocol I've used the buffer was used to remove the libraries from the beads after...
Forum: Illumina/Solexa 11-13-2018, 06:02 AM
Replies: 4
Views: 1,571
Posted By GSviral
Hi Gaetan, For sequencing on the MiSeq at...

Hi Gaetan,

For sequencing on the MiSeq at least I know there is a recommendation to use a 5% PhiX spike-in for low diversity libraries (dependent on which version of the MiSeq Control Software you...
Forum: Illumina/Solexa 11-13-2018, 05:56 AM
Replies: 3
Views: 935
Posted By GSviral
Hi, It looks to me that your run is...

Hi,

It looks to me that your run is borderline overclustered from the second image. V2 chemistry recommends a clustering range of 1000 - 1200 K/mm2. This is probably why you are seeing a drop in...
Forum: Illumina/Solexa 10-16-2018, 01:46 AM
Replies: 1
Views: 900
Posted By GSviral
Hi, It's tough to get an idea of what is...

Hi,

It's tough to get an idea of what is happening just by cluster density and loading concentration alone.

- For the 450 K/mm2 run, what were your pass filter and Q-scores looking like...
Forum: Illumina/Solexa 09-06-2018, 11:24 PM
Replies: 3
Views: 1,334
Posted By GSviral
Hi bheida, This is a good question. I may be...

Hi bheida,

This is a good question. I may be a bit rusty on my theory but I can try to help.

I believe when the libraries have hybridised to the flow cell via the P5 and P7 oligos the...
Forum: Illumina/Solexa 08-23-2018, 11:19 PM
Replies: 1
Views: 1,168
Posted By GSviral
Hi James, As far as I'm aware there will not...

Hi James,

As far as I'm aware there will not be any change in the raw data produced by the run after changing the HT to LT. I believe it simply allows the correct indexes to be assigned that were...
Forum: Illumina/Solexa 04-25-2018, 07:05 AM
Replies: 7
Views: 3,395
Posted By GSviral
Hi Greenleaf, I typically always perform...

Hi Greenleaf,

I typically always perform paired-end sequencing to maximise the data output from whichever sequencer I am using. In terms of alignment, I would hazard a guess that paired-end...
Forum: Illumina/Solexa 04-13-2018, 02:07 AM
Replies: 6
Views: 2,954
Posted By GSviral
Marc, A quick look on the product page for...

Marc,

A quick look on the product page for the TruSeq RNA Exome kit states that multiplex options are 24 single index or up to 96 combinatorial dual indexes. This seems like it would meet your...
Forum: Illumina/Solexa 04-13-2018, 01:55 AM
Replies: 6
Views: 2,954
Posted By GSviral
Hi Marc, I can confirm from experience that...

Hi Marc,

I can confirm from experience that the TruSeq RNA Access (now RNA Exome) performs really well with degraded FFPE material and generates consistent, high quality libraries from FFPE...
Forum: Bioinformatics 11-06-2015, 03:44 AM
Replies: 3
Views: 1,748
Posted By GSviral
Hey Romainb, I was able to resolve the...

Hey Romainb,

I was able to resolve the problem yup. What you have to do is:

When you import, for example, you BLAST output file (.xml or .tab etc.) you will have 3 boxes. Your input file will...
Forum: Bioinformatics 10-14-2015, 12:57 AM
Replies: 3
Views: 1,230
Posted By GSviral
Windows Command Prompt FINDSTR

Hello,

I am currently working with the latest BLAST+ package configured to be a local BLAST nt database using Windows command prompt.

I have set up the database to run BLAST which works without...
Forum: Bioinformatics 10-13-2015, 04:42 AM
Replies: 12
Views: 2,212
Posted By GSviral
Thanks GenoMax. Seems I will have to find an...

Thanks GenoMax.

Seems I will have to find an alternative route.

Sometimes if I am BLASTing a particularly large FASTA file I will get a segmentation fault a short time after the command has...
Forum: Bioinformatics 10-13-2015, 04:26 AM
Replies: 12
Views: 2,212
Posted By GSviral
Thanks for the help GenoMax. I may just have to...

Thanks for the help GenoMax. I may just have to find a different computer to do this all on unfortunately.

Just as a revision, in case I have installed something incorrectly.

I downloaded the...
Forum: Bioinformatics 10-13-2015, 04:02 AM
Replies: 12
Views: 2,212
Posted By GSviral
GenoMax, Yep, I am using the latest BLAST+...

GenoMax,

Yep, I am using the latest BLAST+ package - 2.2.31 along with the most recent nt database.

Could it be possible an OS update could fix the problem?

And yes ha, we are using a 10...
Forum: Bioinformatics 10-13-2015, 03:36 AM
Replies: 12
Views: 2,212
Posted By GSviral
GenoMax, I am using GNOME CentOS 2.16.0. ...

GenoMax,

I am using GNOME CentOS 2.16.0.

Outdated I am sure but my institution are picky about software unfortunately.
Forum: Bioinformatics 10-13-2015, 03:28 AM
Replies: 8
Views: 2,110
Posted By GSviral
Sorry to dig up an old thread. I am having...

Sorry to dig up an old thread.

I am having the same problem with the makeblastdb command. I Get a segmentation fault error even when I type makeblastdb -help. It's like the command doesn't want to...
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