Can I demultiplex run based on i5 sequence?
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18 months ago
bioinfo ▴ 150

Hello,

I was asked to demultiplex a run where the samples had a unique i5 sequence but the i7 sequence was the same for all of them. I have tried to demultiplex it using several --use-bases-mask parameters but I cannot get it to work.

I tried to use --use-bases-mask Y*,n*,I8n*,Y* and a samplesheet that looked like this:

Lane,Sample_ID,Sample_Name,Sample_Plate,Sample_Well,I7_Index_ID,index,I5_Index_ID,index2,Sample_Project,Description

4,Sample1,Sample1,,,,,,CGAGTATA,project_name

I also tried to use --use-bases-mask Y*,I8n*,I8n*,Y* and a samplesheet that looked like this:

Lane,Sample_ID,Sample_Name,Sample_Plate,Sample_Well,I7_Index_ID,index,I5_Index_ID,index2,Sample_Project,Description

4,Sample1,Sample1,,,,NNNNNNNN,,CGAGTATA,project_name

None of these options worked for me. Is there a way to demultiplex that data?

Thank you

demultiplexing bcl2fastq • 1.3k views
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18 months ago
GenoMax 142k

You can demultiplex by second index as long as the combinations of i7 and i5 are unique. Why are you trying to omit the first index?

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I tried to omit it the first time because I was not told that it was dual indexed. The group has not given me their i7 indexes. If I ask for them and replace the NNNNNNNN with the sequence and use --use-bases-mask Y,I8n,I8n,Y would the demultiplexing work?

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Correct. It will work.

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Thank you for the help

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Apparently they had not added the i7 indexes to the samples. Is there still a way to demultiplex it?

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What do the indexes look like? There may be a dummy "TCTTTCCC" type sequence present in place of i7 indexes. You can simply add that in as i7 index even though it is not real.

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Would the fragments attach to the flowcell if no p7 was added at all?

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Is no sequence showing up if you simply use a dummy samplesheet with nothing in it so everything goes to "Undetermined"?

If the library was not properly made the run would have failed.

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Would the run have failed even though there were 50 samples on that lane but only 6 were made without the p7? I could demultiplex the rest of the samples without any issue.

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The run will not fail. Those 6 samples would not cluster thus will not be sequenced. Rest of the samples should be fine as long as they have proper adapters.

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