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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 01-15-2013, 10:20 AM   #1
pmiguel
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Lightbulb How to mix single index and dual index TruSeq libraries

In principle I see no issues with mixing dual and single index TruSeq libraries in the same run. The result will be that for the i7 read, the normal single-index sequence will be collected along with an extra 2 bases. For indexes 1-12, these last 2 bases will always be "AT".

I think their i7 sequences (as would be entered in the sample sheet would be as follows) :

>TruSeq_Single_Index_1
ATCACGAT
>TruSeq_Single_Index_2
CGATGTAT
>TruSeq_Single_Index_3
TTAGGCAT
>TruSeq_Single_Index_4
TGACCAAT
>TruSeq_Single_Index_5
ACAGTGAT
>TruSeq_Single_Index_6
GCCAATAT
>TruSeq_Single_Index_7
CAGATCAT
>TruSeq_Single_Index_8
ACTTGAAT
>TruSeq_Single_Index_9
GATCAGAT
>TruSeq_Single_Index_10
TAGCTTAT
>TruSeq_Single_Index_11
GGCTACAT
>TruSeq_Single_Index_12
CTTGTAAT
>TruSeq_Single_Index_13
AGTCAACA
>TruSeq_Single_Index_14
AGTTCCGT
>TruSeq_Single_Index_15
ATGTCAGA
>TruSeq_Single_Index_16
CCGTCCCG
>TruSeq_Single_Index_18
GTCCGCAC
>TruSeq_Single_Index_19
GTGAAACG
>TruSeq_Single_Index_20
GTGGCCTT
>TruSeq_Single_Index_21
GTTTCGGA
>TruSeq_Single_Index_22
CGTACGTA
>TruSeq_Single_Index_23
GAGTGGAT
>TruSeq_Single_Index_25
ACTGATAT
>TruSeq_Single_Index_27
ATTCCTTT

Then the i5 read will always be "TCTTTCCC" -- as that is the segment of the left adapter that would be addressed by the dual index primer for i5.


I brought this up in a previous thread. Therein, a poster, "Vinz", specified exactly what to do in a manner so succinct I had no idea how to interpret it.

Note: this post contains information partially derived from the Illumina Customer Sequence Letter. Illumina specifies in that letter that they give limited permission to "distribute the sequence information outside your institution or to publish the sequence information in presentations, manuscripts, or publications authored by you, as long as it is accompanied by the following copyright notice:"

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

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Old 01-15-2013, 11:25 AM   #2
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In theory you should be able to set index 2 of the single indexed reads as NNNNNNNN and it should demultiplex just fine. If each of your dual indexed samples have a unique index 1 you can even skip the second index read entirely.
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Old 01-15-2013, 11:34 AM   #3
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Philip,

I assume you are asking if this will work if the analysis is being done by MiSeq/BaseSpace. We never analyze the data on our MiSeq (or using BaseSpace for that matter) so I will only speculate about using CASAVA to analyze the data after the run.

Based on what you have said above it should be possible to de-multiplex this run since the tag pairs should all be unique (but pair would be meaningless for those samples which are single indexed).
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Old 01-15-2013, 11:59 AM   #4
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At this point I am pretty sure it will work and it is obvious how to create a sample sheet that will cause the dual indexed and single indexed libraries to demultiplex.

Illumina tech support initially claimed it was not possible, then when I insisted that it should be, backed off to saying we would have to re-run MSR a second time after hacking runinfo.xml to pull it off. But even that is not necessary and the fix is pretty trivial.

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Old 01-16-2013, 04:09 AM   #5
GenoMax
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Quote:
Originally Posted by pmiguel View Post
At this point I am pretty sure it will work and it is obvious how to create a sample sheet that will cause the dual indexed and single indexed libraries to demultiplex.

Illumina tech support initially claimed it was not possible, then when I insisted that it should be, backed off to saying we would have to re-run MSR a second time after hacking runinfo.xml to pull it off. But even that is not necessary and the fix is pretty trivial.

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Do report back and confirm if it does work. Would save a new thread from being created for this question.
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Old 01-16-2013, 11:20 AM   #6
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Quote:
Originally Posted by GenoMax View Post
Do report back and confirm if it does work. Would save a new thread from being created for this question.
Yes, it works fine.

The run itself was less than stellar, but Rick was able to use CASAVA to demultiplex both the single index and the dual index clusters in a single pass.

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Old 01-17-2013, 03:42 AM   #7
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Quote:
Originally Posted by pmiguel View Post
Yes, it works fine.

The run itself was less than stellar, but Rick was able to use CASAVA to demultiplex both the single index and the dual index clusters in a single pass.

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Was this done by CASAVA analysis after the run or on the MiSeq itself?
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Old 01-17-2013, 04:42 AM   #8
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Quote:
Originally Posted by kcchan View Post
In theory you should be able to set index 2 of the single indexed reads as NNNNNNNN and it should demultiplex just fine. If each of your dual indexed samples have a unique index 1 you can even skip the second index read entirely.
Or "TCTTTCCC" -- that is the sequence that gets read if you do and index2 (i5) read on a TruSeq single index library. (Well, if you are doing PE reads. For SR flowcells, I guess you would get the reverse complement of that because the second index has to be read on the same strand as the first index.)

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Old 01-17-2013, 05:07 AM   #9
pmiguel
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Quote:
Originally Posted by GenoMax View Post
Was this done by CASAVA analysis after the run or on the MiSeq itself?
It was done with CASAVA. But I think it would have worked fine on the MiSeq as well if the correct sample sheet had been provided it.

These were amplicon samples generated based on a design I provided the investigator. I inadvertently designed them using the reverse complement of the desired "A" series dual index i7 sequences. Hence, nothing demultiplexed on the MiSeq.

I think it might be possible to force the MiSeq to repeat the entire analysis using a new sample sheet. But I have not done this before. Have you?

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Old 01-17-2013, 07:19 AM   #10
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We have a pretty new MiSeq and I spend a lot of time changing the sample sheet and telling MSR to rerun the analysis!
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Old 01-17-2013, 07:44 AM   #11
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Quote:
Originally Posted by pmiguel View Post

I think it might be possible to force the MiSeq to repeat the entire analysis using a new sample sheet. But I have not done this before. Have you?

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I have done it only once as an exercise on a PC where I installed MiSeq reporter standalone for testing. I am reasonably certain that you should be able to repeat the analysis on the MiSeq. I suggest making a backup copy of the data folder before trying it
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Old 01-17-2013, 12:11 PM   #12
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Originally Posted by microgirl123 View Post
We have a pretty new MiSeq and I spend a lot of time changing the sample sheet and telling MSR to rerun the analysis!
Want to give a quick run down of how you do that? Would be appreciated...

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Old 01-17-2013, 04:49 PM   #13
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It's quite easy to edit a sample sheet in MSR. Fire up Internet Explorer on the MiSeq and go to localhost:8042/ In the Analyses tab, select the run you want to edit. Then go to the Sample Sheet tab and edit the file like you would in Excel. Here you can change the workflow, sample names, adapter sequences, and index sequences. When you're done just click "Save and Requeue" and let the MiSeq crunch the data!
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Old 01-18-2013, 05:00 AM   #14
pmiguel
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Quote:
Originally Posted by kcchan View Post
It's quite easy to edit a sample sheet in MSR. Fire up Internet Explorer on the MiSeq and go to localhost:8042/ In the Analyses tab, select the run you want to edit. Then go to the Sample Sheet tab and edit the file like you would in Excel. Here you can change the workflow, sample names, adapter sequences, and index sequences. When you're done just click "Save and Requeue" and let the MiSeq crunch the data!
Yes, that does sound easy. However when I browse to that URL, the Analyses tab is is empty.

I checked and the directory it is pointing to does appear to be the correct one. Any ideas?

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Old 01-18-2013, 05:06 AM   #15
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Quote:
Originally Posted by pmiguel View Post
Yes, that does sound easy. However when I browse to that URL, the Analyses tab is is empty.

I checked and the directory it is pointing to does appear to be the correct one. Any ideas?

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Did you try looking at a previous run (since you had said that the most recent run was analyzed off-line by CASAVA)?
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Old 01-18-2013, 05:25 AM   #16
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Quote:
Originally Posted by GenoMax View Post
Did you try looking at a previous run (since you had said that the most recent run was analyzed off-line by CASAVA)?
There are no runs at all listed in the Analyses tab. There should be 5, I think -- at least that is the number of runs in that folder if I look at the file system.

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Old 01-18-2013, 05:55 AM   #17
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Firing up IE opened up the "isis viewer" automatically. Once I clicked on the analysis tab lots of previous runs were listed, with the "re-queue" option.

BTW: Illumina released a new update for the MCS software (v.2.1.x) yesterday.
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Old 01-18-2013, 06:24 AM   #18
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Yes, if any runs were displayed in isus view I am confident I could "re-queue" them. Obviously there is a missing file, or something is mis-configured. Guess I could contact tech support...
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Old 01-18-2013, 06:30 AM   #19
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Okay, maybe the information I need would be:

MiSeq Reporter v2.1 Theory of Operation.

You will need to have a myIllumina account to view...

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Old 01-18-2013, 07:44 AM   #20
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Okay, I got it. I needed to delete the "queuedforanalysis.txt" file from the run folder. Then it will immediately begin processing. (I had put the new sample sheet in the run folder.)
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