"Unravelling intratumoral heterogeneity through high-sensitivity single-cell mutational analysis and parallel RNA-sequencing"
Main article:
Rodriguez-Meira, A., Buck, G., Clark, S.-A., Povinelli, B.J., Alcolea, V., Louka, E., McGowan, S., Hamblin, A., Sousos, N., Barkas, N., et al. (2018). Unraveling intratumoral heterogeneity through high-sensitivity single-cell mutational analysis and parallel RNA-sequencing. Molecular Cell.
https://doi.org/10.1016/j.molcel.2019.01.009
Protocol:
Rodriguez-Meira, A., O'Sullivan, J., Rahman, H., Mead., AJ. "TARGET-Seq: A Protocol For High-Sensitivity Single-Cell Mutational Analysis and Parallel RNA Sequencing"
This pipeline provides an integrated framework for the analysis of single-cell targeted sequencing data obtained using TARGET-seq method. The pipeline processes raw fastq files (which can be gzip compressed) or aligned and sorted bam files, and outputs counts tables for every mutation assessed.
Authors: Alba Rodriguez-Meira, Simon McGowan.
You can find example scripts to process TARGET-seq whole transcriptome data in https://github.com/albarmeira/TARGET-seq-WTA/
Processed targeted-single cell genotyping data can be downloaded from this page (3TARGETseq_processed_genotype_calls_legend; 3TARGETseq_processed_genotype_calls).
SCpipeline is freely available under a GPL3 license.
Pre-processing step
If you are using the high-throughput barcoding method described in "TARGET-Seq: A Protocol For High-Sensitivity Single-Cell Mutational Analysis and Parallel RNA Sequencing", start by demultiplexing the sequencing run using:
GenoDemux_Fastq.sh
This will create two fastq files (R1 and R2) per cell. It requires a sample sheet (see example SampleSheet_byWell_TARGET.csv; same for every run) and a metadata spreadsheet specifying the barcodes assigned to each single cell (see example metadata_200325_NB501183.txt; unique per sequencing run). Then proceed to run the script SCgenotype.pl.
This step requires bcl2fastq, fastq-tools and fastq-pair.
Start here once you have individial fastq files per single cell.
To run the pipeline, open the terminal and type:
perl SCgenotype.pl -c SCpipe.conf
The pipeline requires python version 2.7, STAR version 2.4.2a and samtools version 1.1.
Directories and input files are specified through SCpipe.conf file:
FASTQ_DIR </pathto/fastqdirectory/>
Replace </pathto/fastqdirectory/> with the path to directory in which all fastq files to be analyzed are stored
FASTQ_FWD_SUFFIX <_LXX_RX_.fastq.gz>
Replace <_LXX_R1.fastq.gz> with the suffix of read 1 files; in the case of MiSeq platform this is _L001_R1_001.fastq.gz
FASTQ_REV_SUFFIX <_LXX_RX_.fastq.gz>
Replace <_LXX_R2.fastq.gz> with the suffix of read 2 files; in the case of MiSeq platform this is _L001_R2_001.fastq.gz
gPRIMERS_BED </pathto/gPRIMERS.bed>
Replace </pathto/gPRIMERS.bed> with the path to the bed file containing chromosomal coordinates for each primer used to target genomic DNA amplicons. An example file used for analysis of 3'-TARGETseq dataset can be downloaded from this page (See "gPRIMERS.bed").
mPRIMERS_BED </pathto/mPRIMERS.bed>
Replace </pathto/mPRIMERS.bed> with the path to bed file containing chromosomal coordinates for each primer used to target genomic DNA amplicons. An example file used for analysis of 3'-TARGETseq dataset can be downloaded from this page (See "mPRIMERS.bed").
VARIANTS </pathto/variants.tsv>
Replace </pathto/variants.tsv> with a tsv file containing coordinates of each point mutation/indel to be assessed. Start and end coordinates should be the same for all mutations, including indels. Position of insertions and deletions should be - 1bp the annotated position (identified through a bulk sequencing mutational calling pipeline). For example, ASXL1 c.2728_2729del, annotated in chr20:31023242, should be introduced in the variants.tsv file as chr20:31023241 (annotated position minus 1 base). An example file used for analysis of 3'-TARGETseq dataset can be downloaded from this page (See "variants.tsv").
ANALYSIS_DIR </pathto/outdir/>
Replace </pathto/outdir/> with the path to the directory where output count files should be stored. Intermediate directories used for analysis will also be created in here.
STAR_EXECUTABLE </pathto/package/rna-star/2.4.2a/bin/STAR>
Replace </pathto/package/rna-star/2.4.2a/bin/STAR> with path to STAR executable module.
STAR_INDEX_DIR </pathto/Homo_sapiens/UCSC/hg19/Sequence/STAR> Replace </pathto/Homo_sapiens/UCSC/hg19/Sequence/STAR> with path to STAR index directory. In the analysis of TARGET-seq paper, we used hg19.
STAR_NUM_THREADS <number>
Replace with the number of threads to use when running STAR
SAMTOOLS_EXECUTABLE </pathto/package/samtools/1.1/bin/samtools>
Replace </pathto/package/samtools/1.1/bin/samtools> with the path to samtools executable module.
SAMTOOLS_NUM_THREADS <number>
Replace with the number of threads to use when running samtools
SEPARATE_gDNA_SCRIPT </pathto/scripts/genome_reads_gDNA.pl>
Replace </pathto/scripts/genome_reads_gDNA.pl> with the path to genome_reads_gDNA.pl script.
SEPARATE_mRNA_SCRIPT </scripts/genome_reads_mRNA.pl>
Replace </pathto/scripts/genome_reads_mRNA.pl> with the path to genome_reads_mRNA.pl script.
SUMMARISE_SCRIPT </pathto/scripts/getcounts_mpileup_TARGET.py>
Replace </pathto/scripts/getcounts_mpileup_TARGET.py> with the path to getcounts_mpileup_TARGET.py script.