Vignette for Splintr library

WORK IN PROGRESS! VIGNETTE WILL CHANGE FREQUENTLY.

If you used the Splintr library to barcode your cells, follow this vignette to use NextClone.

First thing first:

1. Clone the repository from github: https://github.com/phipsonlab/NextClone.
2. Set the directory/file path in the nextflow.config file for the following parameters:
   1. publish_dir
   2. clone_barcodes_reference
   3. scrnaseq_bam_files

Read the documentations on the homepage, make sure you understand what each of the parameter in the nextclone.config file is for.

Finally read on.

For scRNA-seq data

Before running NextClone, make sure you run cellranger first, and copy out the possorted_genome_bam file in the outs folder.

Creating clone barcode reference file

After you downloaded the FASTA file containing the reference clone barcodes, make sure you convert them to a normal text file where each line denote only the sequence for a clone barcode.

You can do this using a regex replacement. For example, using vim you can do something like this:

%s/>mCHERRY_Barcode_.*\n//g

What this will do is get rid of the name of the barcode and keep only the sequence.

Alternatively, use text editor like VS code or sublime text and use similar regex replacement.

In the future, we may add support for parsing FASTA file as the clone barcode reference.

Setting the parameters

Then make sure at least the following parameters are set in nextflow.config file.

mode = "scRNAseq"
barcode_length = 60
adapter_5prime = "CGATTGACTA"
adapter_3prime = "TGCTAATGCG"

If your clone barcode is not 60 bp, change barcode_length above.

The sequence for the 5’ and 3’ adapter is from the original splintr protocol. If you have asked for some customisation, you probably will have to modify the sequence above. This sequence is determined by whoever made your library. Thus you should ask them for the sequence.

If you do not want to use the 3’ adapter, i.e., you want the barcode to be identified just based on the 5’ adapter, you can set the adater_3prime to "" (empty string). By default, Nextclone will then only use the 5’ adapter to look for the clone barcodes.

Then run main.nf file.

nextflow run main.nf

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