bcl2fastq is interpreting my experiment folder structure incorrectly as MiSeq-style instead of NextSeq-style

Question

[ekofman]

Hello, I am running bcl2fastq and getting the following error:

Unable to find BCL file for 's_1_11101' in: 180320_NB501337_0390_AHLWLCBGX5/Data/Intensities/BaseCalls/L001/C1.1

It is very apparent based on the structure of my experiment directory (and my own outside knowledge, of course) that my single cell data comes from a NextSeq system (bcl2fastq2 Conversion v2.19 - User Guide), which means it should not actually have a level of subdirectories for cycles (C1.1, etc.).

It seems that bcl2fastq is not realizing this and is trying to treat my input as instead having the C-level subdirectories which would be appropriate for a MiSeq or HiSeq 2000/2500 System.

How can I get bcl2fastq to recognize the appropriate experiment folder structure?

Thanks for the assistance.

[h.mon]

What is the command-line you are using?

[ekofman]

bcl2fastq -r 8 -w 8 -p 8 --runfolder-dir 180320_NB501337_0390_AHLWLCBGX5 --output-dir 180320_NB501337_0390_AHLWLCBGX5/Data/Intensities/BaseCalls/ --sample-sheet sample.csv

-r -w and -p are threading parameters

[GenoMax]

It is very apparent based on the structure of my experiment directory (and my own outside knowledge, of course) that my single cell data comes from a NextSeq system (bcl2fastq2 Conversion v2.19 - User Guide), which means it should not actually have a level of subdirectories for cycles (C1.1, etc.).

Even so shouldn't bcl2fastq automatically figure out what kind of sequencer the data is from. It is possible that you are missing some bcl files (happens at times) and would need to use --ignore-missing-bcls option to complete the conversion (those calls would be converted to N's).

Is this 10x data? If so you may want to do the demux using cellranger mkfastq option for that software.

[Devon Ryan]

It's unclear what exactly bcl2fastq looks at to determine the type of machine that produced data. Can you post the directory tree and possible the runInfo.xml somewhere? We can then compare those against our own NextSeq runs to seee what went wrong.

Did the people who did the sequencing not demultiplex the data for you? There's nothing special that needs to be done with 10X data there (you can feed cellranger the normal fastq files produced by bcl2fastq).

[ekofman]

The directory tree looks exactly like the diagram for the NextSeq inputs in the Illumina documentation, and the run info xml looks like this (sorry the formatting is funky, assume all starting tags have appropriate corresponding closing tags):

<\RunInfo xmlns:xsd="http://www.w3.org/2001/XMLSchema" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" Version="4">

<\Run Id="180320_NB501337_0390_AHLWLCBGX5" Number="390">

<\Flowcell>HLWLCBGX5</flowcell>

<\Instrument>NB501337</instrument>

<\Date>180320</date>

<\Reads>

<\Read Number="1" NumCycles="38" IsIndexedRead="N"/>

<\Read Number="2" NumCycles="8" IsIndexedRead="Y"/>

<\Read Number="3" NumCycles="8" IsIndexedRead="Y"/>

<\Read Number="4" NumCycles="38" IsIndexedRead="N"/> </reads>

<\FlowcellLayout LaneCount="4" SurfaceCount="2" SwathCount="3" TileCount="12" SectionPerLane="3" LanePerSection="2">

<\TileSet TileNamingConvention="FiveDigit">

<\Tiles>

<\Tile>1_11101</tile>

... (lots of Tile objects here...)

<\Tile>4_13612</tile>

<\Tile>4_23612</tile>

<\ImageDimensions Width="2592" Height="1944"/> <\ImageChannels> <\Name>Red</name> <\Name>Green</name> </imagechannels> </run> </runinfo>

[GenoMax]

Please use the formatting bar (especially the code option) to present your post better.

Thank you!