bean count

bean count[-samples]: Count (reporter) screen data

bean count-samples (or bean count for a single sample) maps guide into guide counts, allowing for base transition in spacer sequence. When the matched reporter information is provided, it can count the target site edits and alleles produced by each guide. Mapping is efficiently done based on CRISPResso2 modified for base-edit-aware mapping.

bean count-samples \
  --input sample_list.csv   `# sample with lines 'R1_filepath,R2_filepath,sample_name\n'` \
  -b A                      `# base that is being edited (A/G)` \
  -f sgRNA_info_table.csv   `# sgRNA information` \
  -o .                      `# output directory` \
  -r                        `# read edit/allele information from reporter` \
  -t 12                     `# number of threads` \
  --name my_sorting_screen  `# name of this sample run` \

bean count --R1 R1.fq --R2 R2.fq -b A -f sgRNA_info_table.csv -r

By default, bean count[-samples] assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure.

See full detail below.

Input file format

See Input file format for input file formats.

Output file format

count or count-samples produces .h5ad and .xlsx file with guide and per-guide allele counts.

• .h5ad: This output file follows annotated matrix format compatible with AnnData and is based on Screen object in [purturb_tools](https://github.com/pinellolab/perturb-tools). See Data Structure section for more information.

• .xlsx: This output file contains .guides, .samples, .X[_bcmatch,_edits]. (allele_tables are often too large to write into an Excel!)

Full parameters

usage: bean count [-h] --R1 R1 --R2 R2 -b EDITED_BASE -f SGRNA_FILENAME
                  [--guide-start-seq GUIDE_START_SEQ]
                  [--guide-end-seq GUIDE_END_SEQ]
                  [--barcode-start-seq BARCODE_START_SEQ] [-r]
                  [-q MIN_AVERAGE_READ_QUALITY] [-s MIN_SINGLE_BP_QUALITY]
                  [-n NAME] [-o OUTPUT_FOLDER] [-l REPORTER_LENGTH]
                  [--keep-intermediate] [--skip-filtering]
                  [--qstart-R1 QSTART_R1] [--qend-R1 QEND_R1]
                  [--qstart-R2 QSTART_R2] [--qend-R2 QEND_R2]
                  [--gstart-reporter GSTART_REPORTER] [--match-target-pos]
                  [--target-pos-col TARGET_POS_COL] [--guide-bc GUIDE_BC]
                  [--guide-bc-len GUIDE_BC_LEN] [--offset]
                  [--align-fasta ALIGN_FASTA] [--string-allele] [-g] [-m]
                  [--map-duplicated-to-best]
                  [--map-duplicated-hamming-threshold MAP_DUPLICATED_HAMMING_THRESHOLD]
                  [--mask-barcode] [--tiling]

Named Arguments

--R1

FASTQ file path for read 1

--R2

FASTQ file path for read 2.

-b, --edited-base

For base editors, the base that should be ignored when matching the gRNA sequence. For dual editors, feed in comma-separated target base (e.g. A,C)

-f, --sgRNA-filename

sgRNA description file. The format requires three columns: name, sequence, barcode [ reporter [,strand, target_pos], [start_pos, offset] ].

--guide-start-seq

Guide starts after this sequence in R1. The start sequence is located by allowing the base transition from edited_base (If A to G and if C to T).

Default: ''

--guide-end-seq

Guide starts after this sequence in R1

Default: ''

--barcode-start-seq

Barcode + reporter starts after this sequence in R2, denoted as the sense direction (the same sequence direction as R1). The start sequence is located by allowing the base transition from edited_base (If A to G and if C to T).

Default: ''

-r, --count-reporter

Count reporter edits.

Default: False

-q, --min-average-read-quality

Minimum average quality score (phred33) to keep a read

Default: 30

-s, --min-single-bp-quality

Minimum single bp score (phred33) to keep a read

Default: 0

-n, --name

Output name

Default: ''

-o, --output-folder

Default: ''

-l, --reporter-length

length of the reporter

Default: 32

--keep-intermediate

Keep all the intermediate files

Default: False

--skip-filtering

Skip the read filtering based on quality filters

Default: False

--qstart-R1

Start position of the read when filtering for quality score of the read 1

Default: 0

--qend-R1

End position of the read when filtering for quality score of the read 1

Default: 47

--qstart-R2

Same as qstart_R1, for read 2 fastq file

Default: 0

--qend-R2

Same as qstart_R2, for read 2 fastq file

Default: 36

--gstart-reporter

Start position of the guide sequence in the reporter

Default: 6

--match-target-pos

Count the edit in the exact target position.

Default: False

--target-pos-col

Column name specifying the relative target position within reporter sequence.

Default: 'target_pos'

--guide-bc

Construct has guide barcode

Default: True

--guide-bc-len

Guide barcode sequence length at the beginning of the R2

Default: 4

--offset

Guide file has offest column that will be added to the relative position of reporters.

Default: False

--align-fasta

gRNA is aligned to this sequence to infer the offset. Can be used when the exact offset is not provided.

Default: ''

--string-allele

Store allele as quality filtered string instead of Allele object

Default: False

-g, --count-guide-edits

count the self editing of guides

Default: False

-m, --count-guide-reporter-alleles

count the matched allele of guide and reporter edit

Default: False

--map-duplicated-to-best

When found duplicated mapping allowing for intended edits, map them to the best-matching reads

Default: False

--map-duplicated-hamming-threshold

When found duplicated mapping allowing for intended edits, map them to the best-matching reads only when hamming distance is less than 5*value*guide_length for intended edit

Default: 0.1

--mask-barcode

Allow intended base edit in the barcode sequence.

Default: False

--tiling

Specify that the guide library is tiling library without ‘n guides per target’ design

Default: False