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-   -   Change standard index sequence in sample sheet for Miseq (http://seqanswers.com/forums/showthread.php?t=92824)

Tom2013 02-03-2020 04:14 PM

Change standard index sequence in sample sheet for Miseq
 
We synthesized fusion primers (i.e p7(or p5)-index-Transposase- gene_specific_Primer) using the Nextera adapter and index sequences, and prepared a library for Miseq. When I prepared the sample sheet file for Miseq run, I noticed we used wrong sequence for only one index (N707): we used the reverse complementary sequence of N707 (the other index sequences are good).

Now, I may have two options to fix this:
1) use the default index sequences generated by the IEM software in the sample sheet; run Miseq as usual; most samples will be demultiplexed by the Miseq software except a few; then demultiplex the un-demultiplexed sequences for those samples that have used the N707 index.
I still need to figure out how to demultiplex the sequences.

2) After the sample sheet is generated, change the default N707 index sequence to the sequence that was actually synthesized, and then save the sample sheet for Miseq run. Will this work for Miseq software and sequencing?

I prefer option 2), but not sure whether the Miseq run will be successful. Any ideas will be much appreciated.

microgirl123 02-04-2020 10:33 AM

Are you uploading to BaseSpace? If so, you just need to let the analysis finish up and then go in and fix your sample sheet and requeue the analysis under the "more" tab of the summary page. If you're not uploading to BaseSpace it's still possible to fix your sample sheet and re-analyze the data, just a bit more complicated.

Tom2013 02-04-2020 10:44 AM

Quote:

Originally Posted by microgirl123 (Post 230891)
Are you uploading to BaseSpace? If so, you just need to let the analysis finish up and then go in and fix your sample sheet and requeue the analysis under the "more" tab of the summary page. If you're not uploading to BaseSpace it's still possible to fix your sample sheet and re-analyze the data, just a bit more complicated.

No, I am not uploading to BaseSpace. I know re-analyzing data after Miseq run is complicated, as read1 and read2 do not contain barcode sequences.

I wonder whether correcting that index sequence in sample sheet before Miseq run would work. Does change of default index sequence lead to Miseq run or demultiplexing fail?

GenoMax 02-04-2020 03:28 PM

Quote:

Does change of default index sequence lead to Miseq run or demultiplexing fail?
Demultiplexing may fail, if your indexes are non-standard and you provide standard indexes (but the run will not fail so don't worry). Edit the samplesheet and put the actual indexes you used. You should be able to demultiplex the data either locally on MiSeq or using standalone bcl2fastq. If you use the correct samplesheet when you start the run then everything should be fine.

ajthomas 03-25-2020 10:23 AM

Quote:

Originally Posted by GenoMax (Post 230894)
Demultiplexing may fail, if your indexes are non-standard and you provide standard indexes (but the run will not fail so don't worry). Edit the samplesheet and put the actual indexes you used. You should be able to demultiplex the data either locally on MiSeq or using standalone bcl2fastq. If you use the correct samplesheet when you start the run then everything should be fine.

This is probably a bit late for you, but I'll chime in here with my own experience for those who come after. I needed more indexes than were available in any Illumina product. However, rather than make up my own index sequences, I collected all of Illumina's 8 bp indexes, put them together into one list, eliminated any that were duplicates or too similar to others, and renamed them into one sequence. When I use those, I just make the sample sheet in Excel and import it into Local Run Manager to create a run. The system has no problem with nonstandard indexes, or with standard index sequences with a different name.

Tom2013 03-26-2020 08:13 AM

Thanks for your reply. Option 2 worked well.


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