DropRNA

Install baseq_drops

We need python3 and a package called: baseq_drops, which could be installed by:

pip install baseq_drops

After install, you will have a runnable command baseq-Drop

Config file

The pipeline need the following software or resources:

• star: STAR software, for fast alignment of RNA-Seq data;
• samtools: Sorting bam file;
• whitelistDir: The barcode whitelist files for indrop and 10X should be placed under whitelistDir. These files could bed dowmloaded from https://github.com/basedata10/DropRNA/tree/master/whitelist;
• cellranger_ref_<genome>: The key process of read alignment and tagging to genes are inspired and borrowed from the open source cellranger pipeline(https://github.com/10XGenomics/cellranger). The refernces of genome index and transcriptome can be downloaded from https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest. In the config file, the directory of cellrange references is named as cellranger_<genome>.

While running command, the configures are recorded in the file called config_drops.ini:

[Drops]
samtools = /path/to/samtools
star = /path/to/STAR
whitelistDir = /path/to/whitelist_file_directory
cellranger_ref_hg38 = /path/to/reference/refdata-cellranger-GRCh38-1.2.0/

Process Steps

1. Extract the Cell Barcode Counting the number of each kinds of barcode; this will genrate a barcode_count..csv;
2. Cell Barcode correction and filtering Correcting the cell barcode with 1bp mismatch, filtering the barcode with min number of reads;
3. Split the reads of valid Cell Barcodes The raw pair-end raw reads are splitted to 16 single end files for multiprocessing according to the 2bp prefix of barcode; For example, we will get: split..<AA|AT|AC|AG...|GG>.fq
4. Star Alignment Fastq files runs at the same time; The bam file sorted by sequence header is generated;
5. Reads tagging Tagging the reads alignment position to the corresponding gene name
6. Genrating UMI table

Run Command

The main config is:

• --config: config file;
• --genome/-g: genome version;
• --protocol: [10X|indrop|dropseq]
• --minreads: Minimum reads for a barcode
• --name/-n : Sample name
• --fq1/-1: Read 1
• --fq2/-2: Read 2
• --top_million_reads: How many million reads to use, mainly for testing pipeline with fraction of reads
• --dir/-d: output path

If you config the: cellranger_ref_hg38 you can run the following:

baseq-Drop run_pipe --config ./config_drops.ini -g hg38 -p 10X --minreads 10000 -n 10X_test -1 10x_1.1.fq.gz -2 10x.2.fq.gz -d ./