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Search: Posts Made By: kmcarr
Forum: Bioinformatics 03-11-2020, 04:55 AM
Replies: 5
Views: 838
Posted By kmcarr
yy2, The downstream analysis part is a bit...

yy2,

The downstream analysis part is a bit outside my area so I'll have to leave that to others to help you.

Cheers.
Forum: Bioinformatics 03-10-2020, 11:20 AM
Replies: 5
Views: 838
Posted By kmcarr
Sorry, that was an error. I meant to type...

Sorry, that was an error. I meant to type "Performing 3' adapter trimming...."

I have edited my original post to fix this.
Forum: Bioinformatics 03-09-2020, 10:38 AM
Replies: 5
Views: 838
Posted By kmcarr
Rule #1: Do not get hung up on the big red X's in...

Rule #1: Do not get hung up on the big red X's in FastQC.

The thresholds which delineate Pass|Warn|Fail for the various metrics in FastQC were set using beautiful, single species, perfectly random...
Forum: Illumina/Solexa 02-27-2020, 08:01 AM
Replies: 3
Views: 1,053
Posted By kmcarr
mdegraaf, You didn't say how many libraries...

mdegraaf,

You didn't say how many libraries you pooled. If it is more than 12 then it is inevitable that you have more than one library sharing a common i7 index sequence. i7 is the first of the...
Forum: Bioinformatics 02-13-2020, 08:45 AM
Replies: 2
Views: 758
Posted By kmcarr
Sometimes the old solutions are the best...

Sometimes the old solutions are the best solutions, the good ol' Unix join command:

join -e 'NNNNNNNNNNNNNNNNNNNNNNNN' -1 1 -2 1 -a 1 -a 2 -o '0,1.2,2.2' Input1.txt Input2.txt

Assumptions

...
Forum: Sample Prep / Library Generation 11-22-2019, 11:59 AM
Replies: 4
Views: 1,232
Posted By kmcarr
What I am saying is that for those 5 lanes I...

What I am saying is that for those 5 lanes I mentioned there are 2 very closely spaced peaks, one labeled as a peak of ~1,260-1,270bp and the peak just after that labeled as the Upper marker. The...
Forum: Sample Prep / Library Generation 11-22-2019, 08:47 AM
Replies: 4
Views: 1,232
Posted By kmcarr
Use Region Analysis on the TapeStation software...

Use Region Analysis on the TapeStation software instead of Peak Analysis to determine an average size for each sample. Set up a Region from ~150-1000bp on all samples and use that calculate the...
Forum: Sample Prep / Library Generation 11-07-2019, 06:33 AM
Replies: 2
Views: 984
Posted By kmcarr
Don't over interpret the differences between...

Don't over interpret the differences between these two traces since they were obviously performed using different instruments. The one on the left appears to be an Advanced Analytical Fragment...
Forum: Sample Prep / Library Generation 10-16-2019, 08:06 AM
Replies: 6
Views: 1,712
Posted By kmcarr
LV, Your description sounds very familiar....

LV,

Your description sounds very familiar. Our core went through much the same with our Fragment Analyzer: unexplained failures followed by perfect runs, repeated communication with Tech Support...
Forum: Illumina/Solexa 10-10-2019, 04:25 AM
Replies: 2
Views: 1,259
Posted By kmcarr
... (removed pointless follow up)

... (removed pointless follow up)
Forum: Bioinformatics 09-18-2019, 05:53 AM
Replies: 9
Views: 2,804
Posted By kmcarr
By "sum each line" do you mean the total output...

By "sum each line" do you mean the total output per lane? If so then that information is provided directly in the HiSeq output summary before running BCL2FastqQ.
Forum: Sample Prep / Library Generation 09-18-2019, 04:56 AM
Replies: 21
Views: 6,722
Posted By kmcarr
According to Illumina...

According to Illumina (https://www.illumina.com/products/selection-tools/rrna-depletion-selection-guide.html) they have a new product pending for bacteria and plant leaf tissues. They state to...
Forum: Sample Prep / Library Generation 08-02-2019, 07:37 AM
Replies: 2
Views: 1,199
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: 14
Views: 3,496
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: 1,353
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: 1,353
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: 1,353
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: 6
Views: 5,118
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: 1,365
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: 1,626
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: 3,247
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: 15
Views: 9,156
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: 2,174
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: 1,354
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: 1,128
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.
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