Customprimers-SampleSheet error during secondary analysis

[smurugesan]

Hi everyone,

Yesterday i did a 16s MiSeq run using custom primers, when i tried to demultiplex using samplesheet generated through IEM, i found error during secondary analysis to generate Fastq files. I replaced the illumina index sequences with custom index sequences during samplesheet generation. But still I could not overcome the issue. It would be great help, if any one can help to sort this issue. Many thanks in advance.

[pmiguel]

What was the error?

--
Phillip

[smurugesan]

Dear Phillip, Sorry I was not clear in my message. The error was during the demultiplex execution according to the analysis error log. When MSR executes the secondary analysis, the error message was "aborting analysis. Please check error log and sample sheet issues". For your information please find the below link for sample sheet. Thank you.

https://www.dropbox.com/s/6z28l7bx1n9j73r/SampleSheet.csv?dl=0

[pmiguel]

Nothing leaps out at me as being wrong with that sample sheet. You will probably need to take this up with Illumina.

--
Phillip

[pmiguel]

Well, one thing. You mentioned you changed the sample sheet after the run. What sample sheet was used by the MiSeq during the run?

The length of the i7 index read is determined by the length of the sequences in the sample sheet. So if you did not have 12 base indexes in the run sample sheet, then that index read would be whatever length the indexes you did use were. And, if you didn't provide any indexes, then no index read would have been done and it would likely be impossible to demultiplex you run.

--
Phillip

[GenoMax]

Following up @Phillip's suggestion, post this part from your ReadInfo.xml file for this run. It should look something like this

<Read Number="1" NumCycles="151" IsIndexedRead="N"/>
<Read Number="2" NumCycles="8" IsIndexedRead="Y"/>
<Read Number="3" NumCycles="152" IsIndexedRead="N"/>

[thermophile]

Look for WorkflowLog.txt (I demultiplex in basespace where it's in fastqc/files), scroll to the bottom and it will tell you exactly what has caused the sample sheet failure.

[athuyvo]

I'm experiencing a similar problem. Was this resolved?

I ran a MiSeq run with 16s custom libraries/primers and got the same error "abort analysis. Please check error log and sample sheet." No fastq files were produced for my run, but the overall quality of the run was good. I also noticed the base call intensity for the i7 read was pretty low. Could this be a software issue or maybe a primer issue? PhiX was also spiked in at 15%.

[thermophile]

Here's how my header looks for custom amplicon sequencing.

Workflow	GenerateFASTQ
Application	FASTQ Only
Assay	Nextera
Description	
Chemistry	Amplicon

[athuyvo]

Thanks @thermophile. I have same exact format for my sample sheet. I was able to requeue the analysis by essentially ignoring the i7 reads and demultiplex using only i5 barcodes. We think the problem was suboptimal custom i7 primers so the MiSeq couldn't demultiplex using i7 reads.

[thermophile]

you reverse complimented your i7 barcodes right?

[athuyvo]

i7 barcodes were reverse complimented. We were able to salvage our data by mining the adapter reads and found the i7 barcodes.