[10xRNA] dual index demux - Githubissues

epigen-UCSD / epigen_ucsd_django

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[10xRNA] dual index demux #490

Closed biomystery closed 3 years ago

biomystery commented 4 years ago

Run1

The first (HCLTAFX2) has samples that are run using exclusively the new dual index system (SI-TT-XX).

For the first run, I think that would be a good test for demultiplexing using the new cellranger 4.0 with the dual index system.

Run2

The second (HGG7MAFX2) is a mixed run with single and dual index samples (SI-GA and SI-TT).

The second one is more tricky, but would be helpful in the long run, we did a custom bcl2fastq demux with single and dual index separately to get what we needed. Both runs used the run set up R1:28+R2:91+I1:10+I2:10.

https://ucsdepigenomics.slack.com/files/UBN894JTU/F01A0EYQN4X/2020-9-10_frank_test_sheet.xlsx

biomystery commented 3 years ago

For the HCLTAFX2 I used the standard demultiplexing from 10x available with cellranger4.0. For HGG7MAFX2, i did two demultiplexing runs, the first one was the cellranger4.0 demultiplex with the added parameter --filter-dual-index. To get the single index, I used the following custom bcl2fastq

/projects/ps-epigen/software/bcl2fastq/bin/bcl2fastq --use-bases-mask Y28n,I8n,N10,Y90n* \
--run /projects/ps-epigen/nextSeq/200829_NB501692_0034_AHGG7MAFX2 \
--sample-sheet=/home/jbuchanan/20200829_SeqRun_singleIndex_bcl2fastq_custom.csv \
--output-dir /oasis/tscc/scratch/jbuchanan/demux_fastq \
--no-lane-splitting \
--ignore-missing-positions \
--ignore-missing-controls \
--ignore-missing-filter \
--ignore-missing-bcls \
--create-fastq-for-index-reads \

biomystery commented 3 years ago

1243811 Now need justin to enter these seqruns into lims.

biomystery commented 3 years ago

[error]
The samplesheet contains a sample with a single-index (i7 only) sample index,
but the flowcell was run in a dual-index (i7/i5) configuration.  It is not
possible to demultiplex dual-indexed 10x samples and single-indexed 10x samples
within the same run of bcl2fastq. If you wish to proceed, please run mkfastq
with one of the following arguments:
    --filter-dual-index: Only generate a samplesheet with the samples identified
                         by i7/i5 dual-indices (e.g., SI-TT-A6), ignoring single-
                         index samples.  Single-index samples will not be
                         demultiplexed.
    --filter-single-index: Only generate a samplesheet with samples identified
                           by an i7-only sample index, ignoring dual-indexed
                           samples.  Dual-indexed samples will not be
                           demultiplexed.
NOTE: cellranger mkfastq no longer supports demultiplexing Single Cell 3' v1
flowcells. Please use cellranger 3.1 or earlier for that purpose.

biomystery commented 3 years ago

--use-bases-mask Y28n*,I8n*,N10,Y90* > --use-bases-mask Y28n*,I8n*,N10,Y90n*
http://epigenomics.sdsc.edu:8000/nextseq/ZC/285/update/. HCLTAFX2 -> HCHLTAFX2

biomystery commented 3 years ago

https://github.com/epigen-UCSD/utility_epigen/commit/767de17ad94cb7b66f3741b0a7ef418e81e6c0d3