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Forum: Sample Prep / Library Generation

08-19-2020, 08:17 AM

Replies: 12

How to normalize/pool a 16S library when samples are highly variable concentrations??

Views: 4,124

Posted By samd

Hi Grace, This is interesting. What do you...

Hi Grace,

This is interesting. What do you guys use to elute after the last clean up stage? We use water anyway (based off the manufacturers instructions) so I figured adding more water wouldn't...

Forum: Illumina/Solexa

06-22-2020, 01:42 PM

Replies: 7

Anyone using NovaSeq 2x250 for 16S sequencing?? 1 billion + reads..

Views: 2,058

Posted By samd

Yeah very true. We actually moved over to...

Yeah very true. We actually moved over to Ilumina's unique indexes to deal with just that issue. I also know labs that use "staggered" primers which fix that issue as well while also increasing...

Forum: Illumina/Solexa

06-22-2020, 01:00 PM

Replies: 7

Anyone using NovaSeq 2x250 for 16S sequencing?? 1 billion + reads..

Views: 2,058

Posted By samd

Interesting. I was not aware of these issues that...

Interesting. I was not aware of these issues that are unique to NovaSeq.
It sounds like there is a timely need for a Miseq NovaSeq comparison........

Forum: Illumina/Solexa

06-22-2020, 11:27 AM

Replies: 7

Anyone using NovaSeq 2x250 for 16S sequencing?? 1 billion + reads..

Views: 2,058

Posted By samd

Yeah of course for small libraries, the Miseq is...

Yeah of course for small libraries, the Miseq is enough. But we and a lot of other labs are moving towards experiments with huge sample sizes (300+ to even thousands of samples). Having to do...

Forum: Illumina/Solexa

06-19-2020, 01:01 PM

Replies: 7

Anyone using NovaSeq 2x250 for 16S sequencing?? 1 billion + reads..

Views: 2,058

Posted By samd

Anyone using NovaSeq 2x250 for 16S sequencing?? 1 billion + reads..

Title says it all.

This seems like the future for 16S sequencing but haven't heard of anyone validating yet. Seems like 2x250 is enough coverage for 16S and 1 billions plus reads is WAY more cost...

Forum: Illumina/Solexa

06-19-2020, 12:56 PM

Replies: 26

Download BaseSpace runs from an Ubuntu (Linux) box

Views: 12,649

Posted By samd

I actually made a markdown tutorial of how to...

I actually made a markdown tutorial of how to download basespace files through your terminal. Should work on LINUX.
Let me know if it works! :...

Forum: Sample Prep / Library Generation

03-27-2020, 08:56 AM

Replies: 12

How to normalize/pool a 16S library when samples are highly variable concentrations??

Views: 4,124

Posted By samd

@MU Core I actually also have gotten similar...

@MU Core I actually also have gotten similar advice from someone at a sequencing core. The one thing is that I do have huge variability in PCR efficiency and output. However I feel like this...

Forum: Sample Prep / Library Generation

03-23-2020, 06:41 AM

Replies: 12

How to normalize/pool a 16S library when samples are highly variable concentrations??

Views: 4,124

Posted By samd

@nucacidhunter Thank you for this detailed...

@nucacidhunter Thank you for this detailed answer. All these make sense especially the 2 time normalization. Why is lower concentration better? More accurate?
I will employ these modifications once...

Forum: Illumina/Solexa

03-21-2020, 03:04 PM

Replies: 1

Any sequencing cores still open?

Views: 1,488

Posted By samd

Any sequencing cores still open?

Hi,

Anyone know of any trustworthy sequencing cores that are still open for business? Am looking to run on a Miseq.
Thanks,
Sam

Forum: Sample Prep / Library Generation

03-21-2020, 09:09 AM

Replies: 12

How to normalize/pool a 16S library when samples are highly variable concentrations??

Views: 4,124

Posted By samd

How to normalize/pool a 16S library when samples are highly variable concentrations??

Hi all,

I've been having this issue that has been plaguing my PhD. Some of my sequencing runs come back with terrible uneven read coverage. I am guessing it is a pooling problem but still not 100%...

Forum: Illumina/Solexa

11-06-2019, 05:45 PM

Replies: 18

Low data output from questionable sequencing facility

Views: 2,906

Posted By samd

Hi itstrieu, I see. Well I guess I am...

Hi itstrieu,

I see. Well I guess I am getting very low outputs then.. I will run that suggestion by them. It is just strange because my first run at Berkeley which I consider "good" was done at...

Forum: Illumina/Solexa

11-06-2019, 03:21 PM

Replies: 18

Low data output from questionable sequencing facility

Views: 2,906

Posted By samd

Ok so I am guessing the "25M total reads" on...

Ok so I am guessing the "25M total reads" on Basespace actually means 50M since I did PE. Thanks for the suggestion I will look into that.

One thing I just remembered is the QC results were quite...

Forum: Sample Prep / Library Generation

11-06-2019, 03:13 PM

Replies: 2

discrepancy in miseq runs due to sample prep protocol?

Views: 1,612

Posted By samd

discrepancy in miseq runs due to sample prep protocol?

Hi all,

I have had an issue where my first ever Miseq run was great quality: 100k reads per sample with a nice distribution and then after switching sequencing facilities I get lower outputs and...

Forum: Illumina/Solexa

11-06-2019, 01:54 PM

Replies: 18

Low data output from questionable sequencing facility

Views: 2,906

Posted By samd

Hi SNPsaurus, Interesting. I have heard of...

Hi SNPsaurus,

Interesting. I have heard of the spacer primers but I think I am past the point in my PhD of redoing everything. But it would be nice.

This might be a dumb question but I thought...

Forum: Illumina/Solexa

11-06-2019, 01:43 PM

Replies: 18

Low data output from questionable sequencing facility

Views: 2,906

Posted By samd

Hi nucacidhunter, Sorry do you mean manually...

Hi nucacidhunter,

Sorry do you mean manually changing the cluster density to 800k/mm2? Is this something I could tell the facility to do?
And I have been using Ilumina UD indexes so I am guessing...

Forum: Illumina/Solexa

11-06-2019, 01:27 PM

Replies: 18

Low data output from questionable sequencing facility

Views: 2,906

Posted By samd

Hi SNPsaurus, Ok so after discussing with...

Hi SNPsaurus,

Ok so after discussing with them the 11M reads simply referred to the forward reads. The 23M PF refers to the forward and reverse. I see a 40% undetermined reads metric. Which is a...

Forum: Illumina/Solexa

11-06-2019, 09:29 AM

Replies: 18

Low data output from questionable sequencing facility

Views: 2,906

Posted By samd

Hi GenoMax, Under the Indexing QC tab it...

Hi GenoMax,

Under the Indexing QC tab it says Total Reads: 25,058,484 and then PF Reads: 23,419,084. And then the density is at 477. I believe this would be total reads: R1 +R2?
However, when I...

Forum: Illumina/Solexa

11-06-2019, 09:22 AM

Replies: 18

Low data output from questionable sequencing facility

Views: 2,906

Posted By samd

@luc I used the Ilumina unique dual indexes...

@luc

I used the Ilumina unique dual indexes so that should not be an issue right?

@SNPsaurus

I have not done that. I know that is much more accurate we just don't exactly have the...

Forum: Illumina/Solexa

11-06-2019, 09:15 AM

Replies: 18

Low data output from questionable sequencing facility

Views: 2,906

Posted By samd

Hi all, appreciate the responses ...

Hi all,
appreciate the responses

@nucacidhunter
1. Primers are the EMP 515-806 V4 region
2. Cluster density was about 477 if I remember correctly
3. Just 1 library. Usually I was doing 160...

Forum: Illumina/Solexa

11-04-2019, 07:30 PM

Replies: 18

Low data output from questionable sequencing facility

Views: 2,906

Posted By samd

Low data output from questionable sequencing facility

Hi all,

I have been getting some questionable runs from a sequencing facility and was wondering if there could be any clues as to whether it is their fault or my own fault in lab prep.

I am...

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