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Count

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Input files

Experiment File

Comma separated file (CSV) that assigns all fastq files present in a directory to a condidtion and replicate. Each line represents an experiment, which will all be processed in parallel

Condition,Replicate,DNA_BC_F,DNA_UMI,DNA_BC_R,RNA_BC_F,RNA_UMI,RNA_BC_R
Condidtion1,1,C1R1_DNA_barcode_F.fastq.gz,C1R1_DNA_barcode_UMI.fastq.gz,C1R1_DNA_barcode_R.fastq.gz,C1R1_RNA_barcode_F.fastq.gz,C1R1_RNA_barcode_UMI.fastq.gz,C1R1_RNA_barcode_R.fastq.gz
Condidtion1,2,C1R2_DNA_barcode_F.fastq.gz,C1R2_DNA_barcode_UMI.fastq.gz,C1R2_DNA_barcode_R.fastq.gz,C1R2_RNA_barcode_F.fastq.gz,C1R2_RNA_barcode_UMI.fastq.gz,C1R2_RNA_barcode_R.fastq.gz
Condidtion1,3,C1R3_DNA_barcode_F.fastq.gz,C1R3_DNA_barcode_UMI.fastq.gz,C1R3_DNA_barcode_R.fastq.gz,C1R3_RNA_barcode_F.fastq.gz,C1R3_RNA_barcode_UMI.fastq.gz,C1R3_RNA_barcode_R.fastq.gz
Condidtion2,1,C2R1_DNA_barcode_F.fastq.gz,C2R1_DNA_barcode_UMI.fastq.gz,C2R1_DNA_barcode_R.fastq.gz,C2R1_RNA_barcode_F.fastq.gz,C2R1_RNA_barcode_UMI.fastq.gz,C2R1_RNA_barcode_R.fastq.gz
Condidtion2,2,C2R2_DNA_barcode_F.fastq.gz,C2R2_DNA_barcode_UMI.fastq.gz,C2R2_DNA_barcode_R.fastq.gz,C2R2_RNA_barcode_F.fastq.gz,C2R2_RNA_barcode_UMI.fastq.gz,C2R2_RNA_barcode_R.fastq.gz
Condidtion2,3,C2R3_DNA_barcode_F.fastq.gz,C2R3_DNA_barcode_UMI.fastq.gz,C2R3_DNA_barcode_R.fastq.gz,C2R3_RNA_barcode_F.fastq.gz,C2R3_RNA_barcode_UMI.fastq.gz,C2R3_RNA_barcode_R.fastq.gz

Design File

Fasta file of of CRS sequences with unique headers describing each tested sequence

Example file:

>CRS1
GACGGGAACGTTTGAGCGAGATCGAGGATAGGAGGAGCGGA
>CRS2
GGGCTCTCTTATATTAAGGGGGTGTGTGAACGCTCGCGATT
>CRS3
GGCGCGCTTTTTCGAAGAAACCCGCCGGAGAATATAAGGGA
>CRS4
TTAGACCGCCCTTTACCCCGAGAAAACTCAGCTACACACTC

Association Pickle

Python dictionary of CRS to Barcodes

Label File (Optional)

Tab separated file (TSV) of desired labels for each tested sequence

Example file:

CRS1  Positive_Control
CRS2  Negative_Control
CRS3  Test
CRS4  Positive_Control

Count.nf

Options

With --help or --h you can see the help message.

Mandatory arguments:

--dir Fasta directory (must be surrounded with quotes) --e, --experiment-file Experiment csv file --design Fasta of ordered insert sequences. --association Pickle dictionary from library association process.

Optional:

--labels tsv with the oligo pool fasta and a group label (ex: positive_control), a single label will be applied if a file is not specified --outdir The output directory where the results will be saved (default outs) --bc-length Barcode length (default 15) --umi-length UMI length (default 10) --no-umi Use this flag if no UMI is present in the experiment (default with UMI) --merge_intersect Only retain barcodes in RNA and DNA fraction (TRUE/FALSE, default: FALSE) --mpranalyze Only generate MPRAnalyze outputs --thresh minimum number of observed barcodes to retain insert (default 10)

Processes

Processes run by nextflow in the Association Utility. Some Processes will be run only if certain options used and are marked below.

create_BAM or create_BAM_noUMI (if no UMI sequence)

creates a bamfile of barcode and UMI sequences

raw_counts

creates a table of counts for each barcode (where UMIs, if present, are deduplicated)

filter_counts

Remove barcodes that are not the appropriate length

final_counts

Record overrepresended UMIs and final count table

dna_rna_merge_counts or dna_rna_mpranalyze_merge

Merge RNA/DNA count matrices per barcode

final_merge (MPRAnalyze option only)

Merge all DNA/RNA counts into one file

final_label (MPRAnalyze option only)

Label the barcodes

generate_mpranalyze_inputs (MPRAnalyze option only)

Generate inputs for MPRAnalyze, counts tables and annotation tables for rna/dna

dna_rna_merge

Merge each DNA and RNA file label with sequence and insert and normalize

calc_correlations

Calculate correlations between Replicates

make_master_tables

Create tables of each CRS normalized across replicates

Output

The output can be found in the folder defined by the option --outdir. It is structured in folders of the condition as

Files

File tree

outdir
  |-Condition
    |-allreps.tsv
    |-average_allreps.tsv
    |-HepG2_1_2_correlation.txt
    |-HepG2_1_2_DNA_pairwise.png
    |-HepG2_1_2_Ratio_pairwise.png
    |-HepG2_1_2_RNA_pairwise.png
    |-HepG2_all_barcodesPerInsert_box.png
    |-HepG2_barcodesPerInsert.png
        |-Reps
            |-HepG2_1_counts.tsv
            |-HepG2_1_counts.tsv.gz
            |-HepG2_1_DNA_counts.tsv
            |-HepG2_1_DNA_raw_counts.tsv.gz  
            |-HepG2_1_RNA_filtered_counts.tsv.gz
            |-HepG2_1_DNA_filtered_counts.tsv.gz
            |-HepG2_1_RNA_counts.tsv
            |-HepG2_1_RNA_raw_counts.tsv.gz

Files for each Condition

allreps.tsv

TSV of normalized DNA and RNA count, ratio, log2ratio, and number of observed barcodes for each condition, replicate, of every CRS

average_allreps.tsv

mean ratio, log2 ratio, and observed barcodes per condidition normalized for all replicates

HepG2_1_2_correlation.txt

correlation values for a condition and 2 replicates (ie: HepG2 replicate 1 vs replicate 2)

HepG2_1_2_DNA_pairwise.png

Correlation plot of DNA counts condition vs two reps (ie: HepG2 replicate 1 vs replicate 2)

HepG2_1_2_Ratio_pairwise.png

Correlation plot of normalized log2(RNA/DNA) condition vs two reps (ie: HepG2 replicate 1 vs replicate 2)

HepG2_1_2_RNA_pairwise.png

Correlation plot of RNA counts condition vs two reps (ie: HepG2 replicate 1 vs replicate 2)

HepG2_all_barcodesPerInsert_box.png

Box plot of each CRS accross replicates for all barcodes in each condidtion. Colored by the label file.

HepG2_barcodesPerInsert.png

Histogram of number of barcodes detected per CRS

HepG2_group_barcodesPerInsert_box.png

Boxplot of CRS normalized per insert, grouped by labels

Files for each replicate in each condidtion

HepG2_1_counts.tsv

mean ratio, log2 ratio, and observed barcodes per condidition for each replicate

HepG2_1_counts.tsv.gz

table of barcodes with DNA counts and RNA counts

HepG2_1_DNA_counts.tsv

table of barcodes with DNA counts

HepG2_1_DNA_raw_counts.tsv.gz

table of barcodes, UMI, and DNA counts raw

HepG2_1_DNA_filtered_counts.tsv.gz

table of barcodes, UMI, and DNA counts raw, filtered for barcodes of correct length

HepG2_1_RNA_counts.tsv

table of barcodes with RNA counts

HepG2_1_RNA_raw_counts.tsv.gz

table of barcodes, UMI, and RNA counts raw

HepG2_1_RNA_filtered_counts.tsv.gz

table of barcodes, UMI, and DNA counts raw, filtered for barcodes of correct length