CRISPR Count First Run

This tutorial uses the tiny checked workflow fixtures under examples/workflows/fixtures/. It does not download public data. The goal is to show the production assay path, the MAGeCK-compatible count matrix, and the sample QC table in a few commands.

1. Build DotMatch

make

2. Recommended: scaffold or start an assay project

The production path is dotmatch assay new followed by dotmatch assay start (or ./run.sh inside a scaffolded project). That runs preflight check, counts guides, runs CRISPR QC, and writes a reliability report with suggested assay_fixes.tsv edits when thresholds fail.

From the checked fixture:

cd examples/workflows/fixtures
../../dotmatch assay start crispr_assay.toml

To scaffold a fresh project from your own FASTQs:

dotmatch assay new crispr \
  --library guides.csv \
  --reads-dir fastqs/ \
  --out crispr_screen/

cd crispr_screen
./run.sh

Key outputs under the configured out_dir:

• counts.mageck.tsv — MAGeCK-style count matrix

• sample_qc.tsv — per-sample assignment and representation QC

• summary.json — run metadata and assignment rates

• reliability_report.html — evidence-backed preflight/postrun review

• crispr_qc.json — guide-level QC summary

CPU remains the assignment authority. GPU Metal is opt-in via [backend] in the assay spec and requires --metal-validate when enabled.

3. Direct crispr-count (single command)

For a minimal single command without the full assay wrapper:

mkdir -p tmp/crispr-first-run

cat > tmp/crispr-first-run/samples.tsv <<'EOF'
sample_id	fastq
sample_a	examples/workflows/fixtures/sample_a.fastq
sample_b	examples/workflows/fixtures/sample_b.fastq
EOF

crispr-count --samples accepts TSV or CSV sample sheets. With a header, the sample column may be named sample_id, sample, or label; the FASTQ column may be named fastq, fastq_path, reads, path, or file. Without a header, DotMatch treats the first column as the sample name and the second as the FASTQ path.

The fixture library contains three guides:

cat examples/workflows/fixtures/crispr_library.csv

Guide libraries may be TSV or CSV. DotMatch detects common CRISPR headers such as sgRNA, sgRNAID, guide_id, gRNA.sequence, sgRNA_sequence, guide_seq, sequence, Gene, and gene_symbol.

./dotmatch crispr-count \
  --library examples/workflows/fixtures/crispr_library.csv \
  --samples tmp/crispr-first-run/samples.tsv \
  --guide-start 0 \
  --guide-length 4 \
  --k 1 \
  --metric hamming \
  --ambiguity-policy radius \
  --out tmp/crispr-first-run/counts.mageck.tsv \
  --summary tmp/crispr-first-run/qc.json \
  --ambiguous discard

sample_qc.tsv is written automatically beside --out. Progress and QC review warnings go to stderr on long runs.

4. Inspect the count matrix

cat tmp/crispr-first-run/counts.mageck.tsv

Expected output:

sgRNA	Gene	sample_a	sample_b
guide_a	GENEA	0	0
guide_b	GENEB	0	0
guide_c	GENEC	0	1

The output is ready for MAGeCK-style downstream analysis: sgRNA, Gene, then one integer count column per sample. DotMatch does not run MAGeCK statistics; it only writes the count matrix expected by those tools.

5. Inspect sample QC

cat tmp/crispr-first-run/sample_qc.tsv

The key columns are:

• total_reads: input reads observed for the sample.

• assigned_reads: reads assigned uniquely to one guide.

• exact_reads: exact guide-window matches.

• k1_rescued_reads: one-edit rescued reads.

• ambiguous_reads: reads matching multiple guides within the allowed radius.

• no_match_reads: valid guide windows that matched no guide.

• invalid_reads: reads too short for the configured guide window.

• assignment_rate, ambiguous_rate, and no_match_rate: the same outcomes divided by valid extracted reads.

• targets_observed, zero_count_targets, gini_index, and top_1pct_read_fraction: guide-representation checks for quick review.

• candidates_verified: native target candidates checked after indexing.

In sample_a, the fixture deliberately includes one exact read that is withheld because another guide is inside the one-edit radius, one ambiguous one-edit read, one unmatched read, and one invalid short read. That is the behavior DotMatch is designed to expose rather than hide.

6. Verify against the checked fixture outputs

diff -u examples/workflows/fixtures/expected_counts.mageck.tsv \
  tmp/crispr-first-run/counts.mageck.tsv

No diff means the tutorial count matrix matches the repository fixture.

For a public-data CRISPR example, use examples/crispr_guides/run.sh and the checked evidence reports under docs/benchmarks/public_crispr/.