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Mixing dual and single index TruSeq samples in a single MiSeq run pmiguel Illumina/Solexa 1 12-21-2012 05:21 AM
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Old 02-12-2013, 11:18 AM   #21
pmiguel
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By the way, as far as the dual + single indexed libraries demultiplexing via MCS/RTA, it does work:



The first 3 libraries are single index genomic DNA libraries. Also included an amplicon set (generated by a customer) with 96 samples using the "TruSeq Custom Amplicon" dual indexes.

Oh, just in case:

Oligonucleotide sequences 2007-2012 Illumina, Inc. All rights reserved.

The MiSeq happily demultiplexed all 99 index pairs at a >97% efficiency.

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Old 05-27-2014, 01:15 AM   #22
Mosiep
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Is it possible to combine/mix samples together for a single run in 454 sequencing? is it practical ?if so what are the benefits? I am new to 454 sequencing
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Old 05-27-2014, 04:41 AM   #23
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Quote:
Originally Posted by Mosiep View Post
Is it possible to combine/mix samples together for a single run in 454 sequencing? is it practical ?if so what are the benefits? I am new to 454 sequencing
454? This is an Illumina thread.

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Old 10-22-2015, 06:45 AM   #24
Jessica_L
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I'm trying to do something similar. I added a single indexed library (6bp P7 index) into a pool of tru seq amplicon libraries (8bp P7 index) and need to demultiplex. I tried putting two "N"s at the end of the 6bp index (i.e. GATCAGNN) on the sample sheet but that doesn't appear to have demultiplexed correctly. Or should it have and maybe the library failed to cluster? It's entirely possible the library failed library construction (colleague generated the library; it was his first ever).

I'm thinking I'll try demultiplexing via CASAVA when the dataset is done just to be sure I set the indexes correctly in the sample sheet. I don't expect MSR to have much for me analysis-wise to requeue since this was a generate fastq workflow.

Does anyone have any thoughts?

Last edited by Jessica_L; 10-22-2015 at 06:46 AM. Reason: Fixed a typo.
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Old 10-22-2015, 07:06 AM   #25
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Quote:
Originally Posted by Jessica_L View Post
I'm trying to do something similar. I added a single indexed library (6bp P7 index) into a pool of tru seq amplicon libraries (8bp P7 index) and need to demultiplex. I tried putting two "N"s at the end of the 6bp index (i.e. GATCAGNN) on the sample sheet but that doesn't appear to have demultiplexed correctly. Or should it have and maybe the library failed to cluster? It's entirely possible the library failed library construction (colleague generated the library; it was his first ever).

I'm thinking I'll try demultiplexing via CASAVA when the dataset is done just to be sure I set the indexes correctly in the sample sheet. I don't expect MSR to have much for me analysis-wise to requeue since this was a generate fastq workflow.

Does anyone have any thoughts?
I believe it should work if you add "AT" at the end of the 6 bp index and repeat the demultiplexing in MiSeq Reporter.
Or you can leave the index with only the 6 bases (remove the "NN) and demultiplex; this should demultiplex those samples. Then, if needed, you can do another demultiplexing for the amplicon libraries, using 8 bases in the sample sheet.
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Old 10-22-2015, 07:23 AM   #26
Jessica_L
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Quote:
Originally Posted by JBKri View Post
I believe it should work if you add "AT" at the end of the 6 bp index and repeat the demultiplexing in MiSeq Reporter.
Or you can leave the index with only the 6 bases (remove the "NN) and demultiplex; this should demultiplex those samples. Then, if needed, you can do another demultiplexing for the amplicon libraries, using 8 bases in the sample sheet.
Thanks so much-- I'll give it a try!
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Old 05-22-2017, 07:32 AM   #27
JuNimey
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I found this thread since I was searching for sequencing Nextera and TruSeq LT libraries on one lane in a MiSeq. I only need the i7 read for the nextera libraries and the TruSeq libraries I have are modified and contain 8-base barcodes (as the Nextera libraries do as well). Can you give me an advice on what to enter in the sample sheet, so that this will work properly? It's my first time sequencing on a MiSeq and I would appreciate any help! Thanks a lot!
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Old 05-22-2017, 09:05 AM   #28
thermophile
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In IEM, select truseq single index to see how the sheet should be laid out. Then use that header and column names for the sheet you put together
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Old 05-22-2017, 09:07 AM   #29
GenoMax
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For reference. IEM = Illumina experiment manager (windows only).
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Old 05-22-2017, 09:18 AM   #30
thermophile
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oh didn't realize that.

if poster isn't windows, I can search for an example sample sheet
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Old 05-22-2017, 09:22 AM   #31
GenoMax
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Don't worry. I am sure poster can find a windows machine. Or they can use the MiSeq computer.
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Old 05-24-2017, 03:10 AM   #32
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Thanks a lot! I'll see if these information will be enough for my colleagues, who know how to set up the MiSeq
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