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Search: Posts Made By: kmcarr
Forum: Bioinformatics 11-13-2017, 05:38 AM
Replies: 6
Views: 420
Posted By kmcarr
You have the wrong sequences in your adapter...

You have the wrong sequences in your adapter file. The TruSeq Small RNA adapter sequences differ from the standard TruSeq RNA/DNA adapters. Also, you do not need the individual index sequences, just...
Forum: Bioinformatics 10-30-2017, 07:35 AM
Replies: 5
Views: 449
Posted By kmcarr
Have the reads been trimmed?

Have the reads been trimmed?
Forum: Illumina/Solexa 10-03-2017, 06:12 AM
Replies: 2
Views: 515
Posted By kmcarr
This is not directly in answer to your question,...

This is not directly in answer to your question, but is something you need to think about if you are using custom primers and still adding PhiX to your run. If you put the custom primers in the...
Forum: Bioinformatics 09-19-2017, 08:51 AM
Replies: 5
Views: 473
Posted By kmcarr
As suspected the index sequences in the...

As suspected the index sequences in the SampleSheet are in the wrong orientation.


Index1 p7 end primer showing index read (i7) primer annealed

Index (i7)...
Forum: Bioinformatics 09-19-2017, 06:18 AM
Replies: 1
Views: 300
Posted By kmcarr
Ensembl download page...

Ensembl download page (https://www.ensembl.org/info/data/ftp/index.html)

Species table in the middle of page. Type 'Xenopus' in the Filter box (right side of table header). There are links to...
Forum: Bioinformatics 09-19-2017, 06:08 AM
Replies: 5
Views: 473
Posted By kmcarr
BD, First things first; assuming that your...

BD,

First things first; assuming that your libraries are standard Illumina design your barcodes are NOT part of R1. In the standard Illumina library design and run configuration the index read is...
Forum: RNA Sequencing 09-15-2017, 11:06 AM
Replies: 5
Views: 778
Posted By kmcarr
In one sense that is more accurate, but...

In one sense that is more accurate, but calculating coverage like this for RNA-Seq experiments is meaningless since it doesn't take into account the wide variation in transcript abundance. Within a...
Forum: Bioinformatics 09-15-2017, 06:08 AM
Replies: 7
Views: 831
Posted By kmcarr
What about the case where there is more than one...

What about the case where there is more than one sequence which match the longest length? Save all? Only the first or last?
Forum: Illumina/Solexa 09-05-2017, 06:53 AM
Replies: 17
Views: 978
Posted By kmcarr
For any low quality or low quantity RNA samples...

For any low quality or low quantity RNA samples our core does not use TruSeq. We have had good results with the RNA-Seq library kits from NuGEN (http://www.nugen.com/products/rna-seq). For the sample...
Forum: Bioinformatics 08-17-2017, 06:35 AM
Replies: 1
Views: 378
Posted By kmcarr
Unless it is explicitly stated that a reported...

Unless it is explicitly stated that a reported DNA sequence is presented in a 3' to 5' orientation it is accepted convention that all DNA sequences are written 5' to 3'. All sequence data reported by...
Forum: Sample Prep / Library Generation 07-10-2017, 07:12 AM
Replies: 5
Views: 666
Posted By kmcarr
This is VERY important. We did a miRNA-Seq...

This is VERY important. We did a miRNA-Seq project for a client from Drosophila and were not aware of this fact. Output reads were 70-90% 2S rRNA depending on library. There is a specific protocol ...
Forum: Illumina/Solexa 06-08-2017, 05:27 AM
Replies: 9
Views: 1,755
Posted By kmcarr
You can use dual indexes (or dual unique indexes)...

You can use dual indexes (or dual unique indexes) even with single end reads. Here is a link...
Forum: Illumina/Solexa 05-31-2017, 06:51 AM
Replies: 19
Views: 3,092
Posted By kmcarr
In our core we did the following test: Project...

In our core we did the following test: Project with 64 RNA-Seq libraries prepared using Illumina TruSeq Stranded mRNA HT Dual Index kit on a Perkin Elmer Sciclone G3 robot. We also add a second...
Forum: Illumina/Solexa 05-28-2017, 07:07 AM
Replies: 3
Views: 683
Posted By kmcarr
Your DNA is more than adequate for 16S...

Your DNA is more than adequate for 16S metagenomics. 16S libraries are created entirely by PCR so only a tiny amount of input DNA is required. 16S amplicon sequencing is nearly always done with PE250...
Forum: Illumina/Solexa 05-25-2017, 05:18 AM
Replies: 9
Views: 1,755
Posted By kmcarr
I have heard unconfirmed reports that both Kapa...

I have heard unconfirmed reports that both Kapa and Bioo are planning on offering sets of 96 dual-unique indexes.
Forum: Illumina/Solexa 03-28-2017, 01:35 PM
Replies: 4
Views: 899
Posted By kmcarr
Realistic output for HiSeq 4000

The official spec for PF Clusters on the HiSeq 3000/4000 is 270-312M per lane. It seems that it is pretty easy to exceed this specification but I would like to ask regular operators of a 3000/4000...
Forum: Illumina/Solexa 03-15-2017, 08:59 AM
Replies: 6
Views: 1,089
Posted By kmcarr
Don't focus as much on "same reads per sample"...

Don't focus as much on "same reads per sample" but more on "enough reads per sample" even if the sample-to-sample count varies.

When dealing with 100's of samples in a typical metagenomic...
Forum: Illumina/Solexa 03-05-2017, 06:46 AM
Replies: 8
Views: 1,409
Posted By kmcarr
Lots of excellent advice from Number6. We do a...

Lots of excellent advice from Number6. We do a ton of highly multiplexed metagenomic amplicon sequencing in our lab and would add one extra tip for after the final PCR step, before pooling. We use...
Forum: Illumina/Solexa 02-16-2017, 06:37 AM
Replies: 5
Views: 735
Posted By kmcarr
It is not always the case that the PCR primers...

It is not always the case that the PCR primers are part of the read. In the two most cited 16S-V4 protocols (Caporaso & Knight, Kozich & Schloss) custom sequencing primers which match the target...
Forum: Illumina/Solexa 01-16-2017, 11:30 AM
Replies: 3
Views: 879
Posted By kmcarr
Mike, "Solexa" quality encoding of Q+64 has...

Mike,

"Solexa" quality encoding of Q+64 has not been used in several years (eons in Next Generation Sequencing time). Hell, nobody even calls it "Solexa" anymore; it is Illumina. Do exactly what...
Forum: Illumina/Solexa 01-10-2017, 12:19 PM
Replies: 113
Views: 18,117
Posted By kmcarr
Having 4 channels doesn't mean they are separate...

Having 4 channels doesn't mean they are separate lanes in the same sense as the HiSeq. The NextSeq 500/550 flow cells look just like that with 4 channels yet there is but a single input port so the...
Forum: Illumina/Solexa 01-10-2017, 05:51 AM
Replies: 113
Views: 18,117
Posted By kmcarr
@GenoMax: Yeah I agree, I was just feeling...

@GenoMax: Yeah I agree, I was just feeling particularly crabby this AM. I'm also remembering the time we sprung for a second GAIIx only to have Illumina Announce the HiSeq a week or two after that...
Forum: Illumina/Solexa 01-10-2017, 05:19 AM
Replies: 113
Views: 18,117
Posted By kmcarr
Our core just bought a new 4000, and by just, I...

Our core just bought a new 4000, and by just, I mean installed last week. Explains why Illumina was pushing us all throughout December to get the PO submitted.
Forum: Illumina/Solexa 01-08-2017, 04:27 PM
Replies: 23
Views: 6,626
Posted By kmcarr
A correction Geno, %PF does not change. PF...

A correction Geno, %PF does not change. PF clusters are set at cycle 25. Q scores of course are calculsted on each cycle and later cycles always have lower quality than earlier ones.

Shimul,...
Forum: Illumina/Solexa 12-13-2016, 09:40 AM
Replies: 6
Views: 818
Posted By kmcarr
Are you sure the issue is with bcl2fastq? QScores...

Are you sure the issue is with bcl2fastq? QScores are assigned (binned or not) by RTA. bcl2fastq merely extracts the scores already stored in the BCL file by RTA.
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