[GenoMax]

I generally never use the --use-bases-mask so I will defer to HESmith's expertise.

Dropping the --use-bases-mask altogether and using a samplesheet with 7bp indexes should work as well. That should produce three files: R1 - First read, R2- Second tag or whatever is there, R3 - Should be the real read 2. When the edited RunInfo.xml file is used.

[HESmith]

Dropping the --use-bases-mask altogether and using a samplesheet with 7bp indexes should work as well. That should produce three files: R1 - First read, R2- Second tag or whatever is there, R3 - Should be the real read 2.

This will work only if you know what the second index sequence is supposed to be for the single indexed libraries, so that it can be provided in the sample sheet. Admittedly, it should be straightforward to determine, if you understand how the second index read is generated and you have the adapter sequences for the single indexed libraries at hand.

The inability to treat different lanes as different types of libraries is a real limitation of Illumina's approach to demultiplexing. Use-bases-mask functions at the level of the entire flow cell, which necessitates multiple passes at demultiplexing in cases like this one.

(This response assumed an unedited RunInfo.xml; see subsequent reply re: edited RunInfo.xml)

[HESmith]

When the edited RunInfo.xml file is used.

Yes, the edited RunInfo.xml treats the second read as the index (IsIndexedRead="Y"), and all other reads as data (IsIndexedRead="N"). So, R1 becomes read 1 data (101bp), R3 becomes read 2 data (8bp), and R4 becomes read 3 data (101bp). So it would be necessary to delete read 2 and rename read 3.

Seems more straightforward just to employ --use-bases-mask and get the output you want.

[GenoMax]

@HESmith: If you are sure that --use-bases-mask (as you had suggested) is going to work then it is the straightforward option.

That said I went back and re-tested --use-bases-mask Y*,I6n,Y* option. Since read 3 is no longer designated as an index read one does not need the additional n* after I6n. You should get the three read files that we have already talked about.

[emixaM]

So,

I used the original RunInfo.xml and Y101,I6n*,n*, Y101 and --ignore-missing-bcl and I can see :

[2015-02-19 13:54:06]	[r102-n81]	[all]	INFO: all completed successfully.

THANKS A LOT FOR YOUR HELP!