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Forum: Sample Prep / Library Generation 08-02-2019, 07:37 AM
Replies: 2
Views: 270
Posted By kmcarr
Are you sure that it really is low cluster...

Are you sure that it really is low cluster density? Do you have access to the Thumbnails? When a flow cell is very overloaded it may falsely report a low cluster density. I ask because our experience...
Forum: Illumina/Solexa 05-31-2019, 07:48 AM
Replies: 8
Views: 1,312
Posted By kmcarr
This is interesting; do you know the mechanism...

This is interesting; do you know the mechanism this causes this result?
Forum: Illumina/Solexa 05-10-2019, 04:00 AM
Replies: 5
Views: 878
Posted By kmcarr
Yes, Illumina sequencers absolutely require...

Yes, Illumina sequencers absolutely require sequencing and index read* primers. Bridge amplification and sequence generation are separate things. Every Illumina reagent kit/cartridge has tubes/wells...
Forum: Illumina/Solexa 05-09-2019, 01:36 PM
Replies: 5
Views: 878
Posted By kmcarr
The custom sequencing and index primers are...

The custom sequencing and index primers are complementary to the target specific portions of the original PCR primers, plus the pad and linker. For example, if the amplicon you are sequencing is...
Forum: Illumina/Solexa 05-09-2019, 04:35 AM
Replies: 5
Views: 878
Posted By kmcarr
It sounds like your new lab is following the...

It sounds like your new lab is following the protocol developed by Patrick Schloss' lab (or something very similar) for construction dual indexed Illumina amplicon libraries. You can review the full...
Forum: Oxford Nanopore 05-09-2019, 04:27 AM
Replies: 5
Views: 2,491
Posted By kmcarr
Yes you can, which is actually kind of scary. So...

Yes you can, which is actually kind of scary. So long as you have a copy of MinKNOW installed on your local computer, and know the IP of a MinION/GridION computer you can connect remotely and have...
Forum: Illumina/Solexa 05-09-2019, 04:02 AM
Replies: 2
Views: 823
Posted By kmcarr
Phillip, Our lab does essentially the same...

Phillip,

Our lab does essentially the same as you, remove solution from cartridge, mix in Eppendorf, return to cartridge, but they use disposable Pasteur pipets to suck the sample out of and...
Forum: Illumina/Solexa 04-18-2019, 06:06 AM
Replies: 3
Views: 777
Posted By kmcarr
Julia, Pardon what may be a stupid question...

Julia,

Pardon what may be a stupid question but how did you get the run started without a sample sheet? The MiSeq requires a sample sheet matching the reagent cartridge name and if it can't find...
Forum: General 04-03-2019, 01:12 PM
Replies: 2
Views: 1,196
Posted By kmcarr
It looks like you got hold of a very old (in...

It looks like you got hold of a very old (in Illumina time scale) FastQ file. Have a look at the Illumina sequence identifiers...
Forum: General 02-20-2019, 06:29 AM
Replies: 11
Views: 4,427
Posted By kmcarr
Hello all. It has been a while since Illumina...

Hello all. It has been a while since Illumina BaseSpace ClarityLIMS (nee Genologics) has been discussed and I wanted to see how users are feeling about the direction of the product since Illumina has...
Forum: Illumina/Solexa 11-26-2018, 11:59 AM
Replies: 7
Views: 1,468
Posted By kmcarr
In my opinion it is unwise to install software...

In my opinion it is unwise to install software patches which have not been tested for compatibility by Illumina. We do not patch any instrument computers with anything other than patches/upgrades...
Forum: Bioinformatics 11-14-2018, 04:35 AM
Replies: 4
Views: 942
Posted By kmcarr
frymor, You correctly identified the cause...

frymor,

You correctly identified the cause in your last statement above. The Illumina NextSeq500 (and NovaSeq) use 2 Color Chemistry for tagging and identifying bases. Using this system G's are...
Forum: Sample Prep / Library Generation 11-01-2018, 04:20 AM
Replies: 4
Views: 882
Posted By kmcarr
I have only ever used the BluePippin so can't...

I have only ever used the BluePippin so can't make a comparison. I can say that I regularly isolated 200bp fragments using the BluePippin and was more than satisfied with the results.
Forum: Sample Prep / Library Generation 10-31-2018, 10:08 AM
Replies: 4
Views: 882
Posted By kmcarr
We purchased a BluePippin long ago and it has...

We purchased a BluePippin long ago and it has always had the ability to resolve both long and short fragments, dependent upon which gel cassette and program was run. What the BluePippin has that the...
Forum: Illumina/Solexa 10-31-2018, 10:01 AM
Replies: 1
Views: 677
Posted By kmcarr
Yes. An 'N'. Other than that no.

Yes. An 'N'. Other than that no.
Forum: Bioinformatics 09-14-2018, 05:11 AM
Replies: 1
Views: 783
Posted By kmcarr
Robert, No, there is no additional...

Robert,

No, there is no additional filtering. The %PF column is telling you what percentage of the original (Raw) clusters those reported PF clusters represent. You could back calculate from the...
Forum: Bioinformatics 08-27-2018, 05:24 AM
Replies: 1
Views: 813
Posted By kmcarr
Jeff, Always pre-process read data before...

Jeff,

Always pre-process read data before downstream analysis. This includes adapter trimming and quality filtering even if you don't expect to find much if any adapter.

As an aside you should...
Forum: Bioinformatics 08-06-2018, 05:51 AM
Replies: 2
Views: 800
Posted By kmcarr
This result indicates that your library included...

This result indicates that your library included a large percentage of fragments with insert size less than or equal to 200bp in length. The way Trimmomatic paired end trimming operates (by default)...
Forum: General 06-13-2018, 06:10 AM
Replies: 1
Views: 1,651
Posted By kmcarr
Based on the completely non-rigorous analysis of...

Based on the completely non-rigorous analysis of project submissions to our core I would say that 16S (and other amplicon) sequencing is nowhere near dead.
Forum: Illumina/Solexa 05-24-2018, 04:40 AM
Replies: 4
Views: 1,342
Posted By kmcarr
Not surprising for bacterial RNA-Seq. Bacteria...

Not surprising for bacterial RNA-Seq. Bacteria will transcribe large tracks of their genome from both strands, in polycistronic primary transcripts which may overlap. The implication is that reads...
Forum: Sample Prep / Library Generation 05-11-2018, 07:31 AM
Replies: 5
Views: 1,424
Posted By kmcarr
microgirl, What plate reader are you using?

microgirl,

What plate reader are you using?
Forum: Bioinformatics 05-10-2018, 04:18 AM
Replies: 1
Views: 521
Posted By kmcarr
The option "-ubam" directs intersectBed to write...

The option "-ubam" directs intersectBed to write the output as uncompressed BAM. Since your input was undoubtedly compressed it's not surprising that the uncompressed output is larger even if it...
Forum: 454 Pyrosequencing 04-23-2018, 09:43 AM
Replies: 5
Views: 7,692
Posted By kmcarr
Wow, time to fire up the WayBack Machine Mr....

Wow, time to fire up the WayBack Machine Mr. Peabody.

Yes, 454 "Paired End" technology is similar in design to Illumina Mate Pair tech. A large fragment is circularized with a linker, the circular...
Forum: Illumina/Solexa 03-12-2018, 06:13 AM
Replies: 7
Views: 1,668
Posted By kmcarr
Here's another doc from Illumina...

Here's another doc from Illumina (https://www.illumina.com/content/dam/illumina-marketing/documents/products/techspotlights/cmos-tech-note-770-2013-054.pdf) which also describes the CMOS flow cell...
Forum: Bioinformatics 03-06-2018, 07:15 AM
Replies: 6
Views: 1,398
Posted By kmcarr
Hello lac302, I have attached a perl script...

Hello lac302,

I have attached a perl script I use for this purpose, however when dealing with NextSeq run data the stats are still divided by lane. The script reads the DemultiplexingStats.xml and...
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