Last updated: 2019-07-30

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Knit directory: scRNA-seq-workshop-Fall-2019/

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From sequencer to `cellranger`

In this section, I will show you how to prepare the fastq files and count the scRNAseq matrix by cellranger. After sequencing, one usually gets a folder from the sequencing core with a folder structure like:

The bcl (Binary Base Call) files in the Data folder contains the raw data generated from the illumina sequencers. cellranger wraps the illumina bcf2fastq command into cellranger mkfastq to convert it to fastq files for single-cell RNAseq data.

cellranger mkfastq

For details, check the tutorial from 10x Genoimcs.

On Odyssey computing cluster:

module load bcl2fastq2
cellranger mkfastq --id=test \
                   --run=/path/to/the/run/folder \
                   --csv=test.csv \
                   --jobmode=local \
                   --localmem=40 \
                   --localcores=12

test.csv is a comma seprated file with three columns:

Lane,Sample,Index
1,test_sample,SI-GA-A3

cellranger count

After cellranger mkfastq, we are ready to align the fastqs to the reference genome and count how many reads per gene per cell. These steps are wraped in cellranger count command.

cellranger count --id=sample345 \
                   --transcriptome=/opt/refdata-cellranger-GRCh38-3.0.0 \
                   --fastqs=/home/test/outs/fastq_path/HAWT7ADXX/test_sample/ \
                   --sample=mysample \
                   --expect-cells=6000

What does the output of cellranger count look like?

In the sample345 folder there is an outs folder, and you will find the files Seurat works with in the filtered_feature_bc_matrix folder. There are 3 files in the folder:

ls -sh filtered_feature_bc_matrix/
total 90M
 60K barcodes.tsv.gz  300K features.tsv.gz   90M matrix.mtx.gz
 
# The `barcodes.tsv.gz` contains the cell barcode that passed the `cellranger` filter.
zcat barcodes.tsv.gz | head -5
AAACCCAAGCGCCCAT-1
AAACCCAAGGTTCCGC-1
AAACCCACAGAGTTGG-1
AAACCCACAGGTATGG-1
AAACCCACATAGTCAC-1

# how many cells (barcodes)?
zcat barcodes.tsv.gz | wc -l
11769

# The `features.tsv.gz` contains the ENSEMBLE id and gene symbol
zcat features.tsv.gz | head -5
ENSG00000243485 MIR1302-2HG     Gene Expression
ENSG00000237613 FAM138A Gene Expression
ENSG00000186092 OR4F5   Gene Expression
ENSG00000238009 AL627309.1      Gene Expression
ENSG00000239945 AL627309.3      Gene Expression

## how many genes?
zcat features.tsv.gz | wc -l
33538


# matrix.mtx.gz is a sparse matrix which contains the non-zero counts
zcat matrix.mtx.gz | head -10
%%MatrixMarket matrix coordinate integer general
%metadata_json: {"format_version": 2, "software_version": "3.0.0"}
33538 11769 24825783
33509 1 1
33506 1 4
33504 1 2
33503 1 10
33502 1 5
33500 1 20
33499 1 9

Most of the entries in the final gene x cell count matrix are zeros. Sparse matrix efficiently save the disk space by only recording the non-zero entries.

You see the dimension of the matrix is 33538 x 11769 and the number of non-zero entries is 24825783

e.g. for the subsequent two rows in the sparse matrix:

33509 1 is the index of the row (gene) and column(cell) of that non-zero entry in the matrix, and 1 is the count number.

33506 1 is the index of the row and column of that non-zero entry in the matrix, and 4 is the count number.

From SRA to fastq

Alternatives to `cellranger`

cellranger is very slow. It can take several days to run a mouse single-cell RNAseq data set with even 20 CPUs. There are other tools which can process single-cell RNAseq data set much faster and accurate as well.

Alevin

Kallisto/bustool

sessionInfo()

R version 3.5.1 (2018-07-02)
Platform: x86_64-apple-darwin15.6.0 (64-bit)
Running under: macOS High Sierra 10.13.6

Matrix products: default
BLAS: /Library/Frameworks/R.framework/Versions/3.5/Resources/lib/libRblas.0.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/3.5/Resources/lib/libRlapack.dylib

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

loaded via a namespace (and not attached):
 [1] workflowr_1.4.0 Rcpp_1.0.0      digest_0.6.18   rprojroot_1.3-2
 [5] backports_1.1.3 git2r_0.23.0    magrittr_1.5    evaluate_0.12  
 [9] stringi_1.2.4   fs_1.2.6        whisker_0.3-2   rmarkdown_1.11 
[13] tools_3.5.1     stringr_1.3.1   glue_1.3.0      xfun_0.4       
[17] yaml_2.2.0      compiler_3.5.1  htmltools_0.3.6 knitr_1.21

Part 0

From sequencer to cellranger