SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
MiSeq HT run instead of LT (Wrong sample sheet) JamesRA Illumina/Solexa 1 08-23-2018 11:19 PM
Miseq: ERROR: FlowCell ID is inconsistent across Sample Sheet lines. harijay Illumina/Solexa 1 02-13-2015 04:12 PM
Using MiSeq with both standard and custom sequencing/index primers at the same time? kevin_bryant Illumina/Solexa 1 02-27-2014 12:57 PM
MiSeq Index Primer Sequence krispy Illumina/Solexa 3 12-26-2013 02:31 AM
Free app. for sample tracking and sample sheet generation aubreeh Illumina/Solexa 0 10-15-2013 01:00 PM

Reply
 
Thread Tools
Old 02-03-2020, 04:14 PM   #1
Tom2013
Member
 
Location: Canada

Join Date: Sep 2013
Posts: 19
Default 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.
Tom2013 is offline   Reply With Quote
Old 02-04-2020, 10:33 AM   #2
microgirl123
Senior Member
 
Location: New England

Join Date: Jun 2012
Posts: 199
Default

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.
microgirl123 is offline   Reply With Quote
Old 02-04-2020, 10:44 AM   #3
Tom2013
Member
 
Location: Canada

Join Date: Sep 2013
Posts: 19
Default

Quote:
Originally Posted by microgirl123 View Post
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?
Tom2013 is offline   Reply With Quote
Old 02-04-2020, 03:28 PM   #4
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 7,077
Default

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.
GenoMax is offline   Reply With Quote
Old 03-25-2020, 10:23 AM   #5
ajthomas
Senior Member
 
Location: Utah

Join Date: Mar 2010
Posts: 166
Default

Quote:
Originally Posted by GenoMax View Post
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.
ajthomas is offline   Reply With Quote
Old 03-26-2020, 08:13 AM   #6
Tom2013
Member
 
Location: Canada

Join Date: Sep 2013
Posts: 19
Default

Thanks for your reply. Option 2 worked well.
Tom2013 is offline   Reply With Quote
Reply

Tags
miseq, nextera xt indices

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 02:51 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO