Illumina PIPseq scRNA

Overview

This recipe is for processing Illumina Single Cell 3' RNA Prep kits. The DRAGEN single cell RNA pipeline is adapted to process the unique structure of the PIPseq library.

Example Command Line

• Configure the INPUT options

• Configure the OUTPUT options

• Configure the SCRNA MAP/ALIGN options

• Configure the SCRNA options

We recommend using a linear (non-pangenome) reference for single cell RNA analysis. For more details, refer to Dragen Reference Support.

The following are partial templates that can be used as starting points. Adjust them accordingly for your specific use case.

#!/bin/bash
set -euo pipefail

# Path to DRAGEN hashtable
DRAGEN_HASH_TABLE=<REF_DIR>

# Path to output directory for the DRAGEN run
OUTPUT=<OUT_DIR>

# File prefix for DRAGEN output files
PREFIX=<OUT_PREFIX>

# Define the input sources, either a FASTQ list or FASTQ files.
INPUT_FASTQ_LIST="
  --fastq-list $FASTQ_LIST \
  --fastq-list-sample-id $FASTQ_LIST_SAMPLE_ID \
"

INPUT_FASTQ="
  --fastq-file1 $FASTQ1 \
  --fastq-file2 $FASTQ2 \
  --RGSM $RGSM \
  --RGID $RGID \
"

# Select the input source. Here in this example, we use a INPUT_FASTQ_LIST
INPUT_OPTIONS="
  --ref-dir $DRAGEN_HASH_TABLE \
  $INPUT_FASTQ_LIST \
"

OUTPUT_OPTIONS="
  --output-directory $OUTPUT \
  --output-file-prefix $PREFIX \
"

# RNA alignment requires an annotation file in GTF format.
GTF=<GTF_PATH>

# The single-cell RNA pipeline requires map-align to be true.
# Map-align output can be optionally enabled. Output format options are SAM, BAM, and CRAM (set to BAM here).
SCRNA_MAP_OPTIONS="
  --enable-rna true \
  --enable-map-align true \
  --annotation-file $GTF \
  --enable-map-align-output true \
  --output-format BAM \
"

# Single-cell RNA PIPseq options:

# The PIPseq mode batch option automatically sets the barcode/BI source, the barcode and binning index positions and the barcode sequence list options.
# By default the barcode/BI is read from read 1 and the transcript is obtained from read 2.
# To change the barcode/BI source, use the "--umi-source" option which can be set to read1, read2, qname, or fastq.
# To change the barcode or binning index positions, use "--scrna-barcode-position" and "--scrna-umi-position"
# To change the known barcode sequence list, use "--scrna-barcode-sequence-list"
# See the DRAGEN Single-Cell RNA PIPseq Pipeline User Guide and the DRAGEN Single-Cell RNA User Guide for more details (link below).

# Cell filtering can be done by setting a threshold using either the fixed, ratio, or inflection approaches. 
# Here we set the threshold using the ratio approach.
FILTER_THRESHOLD=ratio

SCRNA_PIPSEQ_OPTIONS="
  --scrna-enable-pipseq-mode true \
  --single-cell-threshold $FILTER_THRESHOLD \
"

# Construct final command line
CMD="
  dragen \
  $INPUT_OPTIONS \
  $OUTPUT_OPTIONS \
  $SCRNA_MAP_OPTIONS \
  $SCRNA_PIPSEQ_OPTIONS
"

# Execute
echo $CMD
bash -c $CMD

For more details on the PIPseq pipeline options, refer to the DRAGEN Single-Cell RNA PIPseq Pipeline User Guide