primer effects before/after introduction of new primers

[cuttlefishh]

New primers were introduced gradually in 2015. Here's what's different:

• Barcodes are now located on the forward (515) primer to enable the usage of various reverse primer constructs to enable longer amplicons (tested on 806r and 926r).
• Degeneracy was added to both the forward and reverse primers (see below), with the intent of removing known biases against Crenarchaeota/Thaumarchaeota (515f modification) and the marine and freshwater Alphaproteobacterial clade SAR11 (806r modification).

The hope is that these new primers improve detection of certain groups. But this complicates comparison of community composition before and after the new primers were introduced. We need to make sure that the conclusions we draw from large meta-analyses are not due to primer differences alone.

[cuttlefishh]

mSystems paper on new primers is here: http://msystems.asm.org/mSystems.00009-15-abstract.php

[thwilkinson]

Regarding the MiSeq run described in the above mSystems paper, using the new 515f/806r primers (barcode residing on the forward primer):

Was the sample sheet for this run constructed any differently from sample sheets used for MiSeq runs with the original primer set?

We have a 16S library where we have incorporated the new primers, and are getting ready to sequence. I am puzzling over whether there is any material difference in preparing the sample sheet, as compared to our previous MiSeq runs with the original primer set.

This might be a trivial request, but seeing an example sample sheet with the new primer set would be most helpful and much appreciated!

[cuttlefishh]

Hi Tom, I think you mean the mapping file, which uses the barcode and linker primer sequences to demultiplex the sequence file for analysis. I sent you an email directly with an example file. Luke

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On May 16, 2017, at 5:48 PM, thwilkinson ***@***.***> wrote: Regarding the MiSeq run described in the above mSystems paper, using the new 515f/806r primers (barcode residing on the forward primer): Was the sample sheet for this run constructed any differently from sample sheets used for MiSeq runs with the original primer set? We have a 16S library where we have incorporated the new primers, and are getting ready to sequence. I am puzzling over whether there is any material difference in preparing the sample sheet, as compared to our previous MiSeq runs with the original primer set. This might be a trivial request, but seeing an example sample sheet with the new primer set would be most helpful and much appreciated! — You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub <#45 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/AFa_ZhVqC4Dc-BHAV6gRD8oQg5YlMOjQks5r6kPggaJpZM4HJsCB>.

[ackermag]

I think I can respond to your request but am unsure of what you mean by sample sheet. Do you mean the list of samples for sequencing?

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On Tue, May 16, 2017 at 5:48 PM, thwilkinson ***@***.***> wrote: Regarding the MiSeq run described in the above mSystems paper, using the new 515f/806r primers (barcode residing on the forward primer): Was the sample sheet for this run constructed any differently from sample sheets used for MiSeq runs with the original primer set? We have a 16S library where we have incorporated the new primers, and are getting ready to sequence. I am puzzling over whether there is any material difference in preparing the sample sheet, as compared to our previous MiSeq runs with the original primer set. This might be a trivial request, but seeing an example sample sheet with the new primer set would be most helpful and much appreciated! — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub <#45 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/AB69gLeqqmC8rixHnia2gFDCELJzNqYpks5r6kPggaJpZM4HJsCB> .

-- Gail Ackermann Knight Lab UCSD [email protected] <[email protected]>

[thwilkinson]

I am referring to the text file that is typically created using the Illumina Experiment Manager (IEM) program, and contains information that the MiSeq instrument needs to perform the sequencing run.

The file also contains a list of samples for sequencing, along with corresponding barcoding indices.

See:
https://support.illumina.com/downloads/miseq_sample_sheet_quick_reference_guide_15028392.html

The IEM software is a good tool for creating sample sheets for sequencing runs of libraries having Illumina indices. However, for libraries with custom, "non-Illumina" barcoding, I didn't see a way to use IEM to create the sample sheet, and so I reach out here. Thank you for your consideration!

[owenssm]

Hi Tom, Attached you’ll find an example sample sheet. We typically modify this sheet instead of using IEM to make it each time. We don’t add all of the barcodes associated with the samples in the sample sheet, but instead add one that is 12bp long that indicates to the MiSeq that it needs to sequence a 12 bp index read. Thanks! Sarah

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----- Sarah M. Owens Biosciences Division Argonne National Laboratory 9700 S. Cass Avenue Bldg. 446, Rm. A176 Lemont, IL 60439 [email protected] 630.252.2101 http://ngs.igsb.anl.gov/ http://scholar.google.com/citations?user=ZCC84OYAAAAJ

On May 17, 2017, at 9:37 AM, thwilkinson ***@***.***> wrote: I am referring to the text file that is typically created using the Illumina Experiment Manager (IEM) program, and contains information that the MiSeq instrument needs to perform the sequencing run. The file also contains a list of samples for sequencing, along with corresponding barcoding indices. See: https://support.illumina.com/downloads/miseq_sample_sheet_quick_reference_guide_15028392.html <https://support.illumina.com/downloads/miseq_sample_sheet_quick_reference_guide_15028392.html> The IEM software is a good tool for creating sample sheets for sequencing runs of libraries having Illumina indices. However, for libraries with custom, "non-Illumina" barcoding, I didn't see a way to use IEM to create the sample sheet, and so I reach out here. Thank you for your consideration! — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub <#45 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/AB_rrVpHL3YF0G1yuZ8sy97KmUyElgbCks5r6wZAgaJpZM4HJsCB>.