Indrop-Flow

Indrops analysis pipeline at BioCore@CRG

The pipeline is based on the DropEST tool: https://github.com/hms-dbmi/dropEst

Installing

1. install docker or singularity.
2. git clone https://github.com/biocorecrg/indrop.git; cd indrop
3. sh INSTALL.sh for checking Nextflow and installing bioNextflow

Running the pipeline

The parameters are listed when using nextflow run indrop.nf --help command.

nextflow run indrop.nf --help
N E X T F L O W  ~  version 19.07.0
Launching `indrop.nf` [gigantic_ride] - revision: 17bd0ef49f
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====================================================
BIOCORE@CRG indropSEQ - N F  ~  version 1.0
====================================================
pairs                         : {PATH}/*R{1,2,3,4}.fastq.gz
genome                        : {PATH}/anno/test.fa.gz
annotation                    : {PATH}/anno/gencode.v28.annotation.gtf
config                        : {PATH}/conf/indrop_v3.xml
barcode_list                  : {PATH}/conf/indrop_v3_barcodes.txt
email                         : yourmail@yourdomain
mtgenes                       : {PATH}/anno/mitoc_genes.txt
version                       : 3_4
library_tag                   : AGATATAA
output (output folder)        : output_v3

You can change them either by using the command line:

nextflow run indrop.nf --pairs "data/{1,2}.fastq.gz" --version 1-2 > log

or changing the params.file You can use the nextflow options for sending the execution in background (-bg) or resuming a failed one (-resume).

nextflow run indrop.nf --pairs "data/{1,2}.fastq.gz" --version 1-2 -bg -resume > log

Indrop versions v1, v2 and v3 are supported

Version 1 and 2

Parameter version: "V1-2"

• File 1: barcode reads. Structure:
  • Cell barcode, part 1
  • Spacer
  • Cell barcode, part 2
  • UMI
• File 2: gene reads

Version 3

Parameter version: "V3_3"

• File 1: cell barcode
• File 2:
  • cell barcode
  • UMI
• File 3: gene read

Parameter version: "V3_4"

• File 1: cell barcode
• File 2:
  • cell barcode
  • UMI
• File 3: gene read
• File 4: library_tag

The parameter library_tag is only needed with version V3_4

Parameters

1. Parameters are specified within the params.config file

The pipeline

1. QC: Run FastQC on raw reads. It stores the results within QC folder.
2. Indexing: It makes the index of the genome by using STAR.
3. dropTag: It creates a "tagged" fastq file with information about the single cell that originated that read in the header.
4. Alignment: It aligns tagged reads to the indexed genome by using STAR. Reasults are stored in Alignments folder.
5. dropEst: It provides the estimation of read counts per gene per single cell. The results are in Estimated_counts folder and consists of an R data object, a file with a list of cells (aka barcode combinations), another with a list of genes and a matrix in Matrix Market format (https://en.wikipedia.org/wiki/Matrix_Market_exchange_formats).
6. dropReport: It reads the R data oject produced by the dropEst step to produce a quality report. It needs a list of mitochondrial genes.
7. multiQC: It wraps the QC from fastQC and STAR mapping in a single output.