Starting with FASTQ files after QC, this pipeline analyses the data to produce a count matrix that represents the predicted number of unique molecules derived from each gene at each spot.
Usage for inference:
Input comprising pair-end fastq files and a whitelist of barcodes for X and Y. Utilising the STAR programme to generate genome indices for the reference file. Our sequence library is pair-end 150bp. Read 2 contains cDNA. For read1, beginning at 5', 1-8 represents barcodeX, 39-46 represents barcodeY, and 47-58 represents UMIs. Both barcodes X and Y contain 50 unique 8bp sequences in the same order. The filename for the output count matrix is ALL_XQ2_sub_2_trim_tagged_dge.txt. The barcode and its coordinates are contained in the xy_index.txt file for further plotting and analysis.
python run_pipeline.py --R1 ALL_XQ2_sub_1.fq.gz \
--R2 ALL_XQ2_sub_2.fq.gz \
--map hg38 \
--bcx lib_barcode_X.txt \
--bcy lib_barcode_Y.txt \
--startx 0 \
--endx 8 \
--starty 38 \
--endy 46 \
--startumi 46 \
--endumi 58
Options for inference:
--map choose the reference from 'hg38' and 'mm10', default = 'mm10'
--thread Specify the number of threads for qc step and dge step. The recommended number is less than 5, default = 3
--R1 input R1 fastq
--R2 input R2 fastq
--bcx input barcode white list for X, default is 'lib_barcode_X.txt'
--bcy input barcode white list for Y, default is 'lib_barcode_Y.txt'
--startx position of barcode X, default is 0
--endx position of barcode X, default is 8
--starty position of barcode Y, default is 38
--endy position of barcode Y, default is 46
--startumi position of umi, default is 46
--endumi position of umi, default is 58
This script requires the following softwares and versions: Python 3.9.7 Nucleotide-Nucleotide BLAST 2.10.1+ STAR 2.7.3a starcode v1.3 samtools 1.9 Drop-seq_tools 2.3.0
Author: Jia Song, Weizhou Qian, Di Sun