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Overview

This is celseq2, a Python framework for generating the UMI count matrix from CEL-Seq2 ¹ sequencing data. We believe data digestion should be automated, and it should be done in a manner not just computational efficient, but also user-friendly and developer-friendly.

Installation

git clone git@github.com:yanailab/celseq2.git
cd celseq2
pip install ./

Quick Start

Running celseq2 pipeline is as easy as 1-2-3. Below is the visualization of the experiment design as same as the sample sheet used in last generation of the pipeline (CEL-Seq-pipeline) as example.

The user had two biological samples which could come from two different experiments, two time-points, two types of tissues, or even two labs. They were denoted as squares and circles, respectively. Each sample had 9 cells.

In principle, what the user would expect as final output was one UMI count matrix for each sample, which meant two UMI matrices in total in this example.

During the CEL-Seq2 experiment, all cells were placed in one 96-well cell plate. They were labeled with same sequencing barcodes (shown as orange plate) but each cell was labeled with its own CEL-Seq2 cell barcode, so that all of them could be sequenced together without losing identities. In details, the nine cells from Experiment-1 were labeled with CEL-Seq2 cell barcodes indexed from 1 to 9, respectively, while the other nine cells from Experiment-2 were labeled with cell barcodes 10 to 18.

Finally the library was distributed in two lanes (purple and dark gray bar) of a sequencer, and got sequenced, which resulted in two sets of CEL-Seq2 data (per lane per sequencing barcode).

What would the pipeline of celseq2 do for the user was to generate UMI-count matrix per experiment with the two sets of CEL-Seq2 data as input.

Step-1: Specify Global Configuration of Workflow

Run new-configuration-file command to initiate configuration file (YAML format), which specifies the details of CEL-Seq2 techniques the users perform, e.g. the cell barcodes sequence dictionary, and transcriptome annotation information for quantifying UMIs, etc.

This configuration can be shared and used more than once as long as user is running pipeline on same species.

new-configuration-file -o /path/to/wonderful_CEL-Seq2_config.yaml

Example of configuration is here.

Example of CEL-Seq2 cell barcodes sequence dictionary is here.

Read "Setup Configuration" for full instructions.

Step-2: Define Experiment Table

Run new-experiment-table command to initiate a table (space/tab separated file format) specifying the experiment layout.

new-experiment-table -o /path/to/wonderful_experiment_table.txt

Fill information into the generated experiment table file row by row.

The content of experiment table in this example could be:

SAMPLE_NAME	CELL_BARCODES_INDEX	R1	R2
wonderful_experiment1	1-9	path/to/lane1-R1.fastq.gz	path/to/lane1-R2.fastq.gz
wonderful_experiment2	10-18	path/to/lane1-R1.fastq.gz	path/to/lane1-R2.fastq.gz
wonderful_experiment1	1-9	path/to/lane2-R1.fastq.gz	path/to/lane2-R2.fastq.gz
wonderful_experiment2	10-18	path/to/lane2-R1.fastq.gz	path/to/lane2-R2.fastq.gz

Read "Experiment Table Specification" for full instructions when more complexed experiment designs take place.

Step-3: Run Pipeline of celseq2

Launch pipeline in the computing node which performs 10 tasks in parallel.

celseq2 --config-file /path/to/wonderful_CEL-Seq2_config.yaml \
    --experiment-table /path/to/wonderful_experiment_table.txt \
    --output-dir /path/to/result_dir \
    -j 10

Read "Launch Pipeline" for full instructions to see how to submit jobs to cluster, or preview how many tasks are going to be scheduled.

Results

All the results are saved under /path/to/result_dir that user specified, which has folder structure:

├── annotation
├── expr                  # <== Here saves all the UMI count matrices
├── input
├── small_diagnose
├── small_fq
├── small_log
├── small_sam
├── small_umi_count
└── small_umi_set

In particular, UMI count matrix for each of the experiments is saved in both CSV and HDF5 format and exported to expr/ folder.

expr/
├── wonderful_experiment1
│   ├── expr.csv          # <== UMI count matrix for cells denoted as squares
│   ├── expr.h5
│   ├── item-1
│   │   ├── expr.csv
│   │   └── expr.h5
│   └── item-3
│       ├── expr.csv
│       └── expr.h5
└── wonderful_experiment2
    ├── expr.csv          # <== UMI count matrix for cells denoted as circles
    ├── expr.h5
    ├── item-2
    │   ├── expr.csv
    │   └── expr.h5
    └── item-4
        ├── expr.csv
        └── expr.h5

Results of item-X are useful to assess variation when FASTQ files from multiple lanes, or technical/biological replicates are present.

Authors

See https://github.com/yanailab/celseq2/blob/master/AUTHORS

License

See https://github.com/yanailab/celseq2/blob/master/LICENSE

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1. Hashimshony, T. et al. CEL-Seq2: sensitive highly- multiplexed single-cell RNA-Seq. Genome Biol. 17, 77 (2016). https://doi.org/10.1186/s13059-016-0938-8 ↩