Lab Evaluation and Handoff

This guide is for a bioinformatics team or core facility evaluating DotMatch on a known-target sequencing assay. It is a local technical evaluation protocol, not a clinical validation protocol and not a substitute for assay-specific controls.

Before processing study data

Confirm all of the following with the assay owner:

  • the target library is the intended revision and contains only the expected fixed-window sequences;

  • the FASTQ read and orientation are known, along with the start and length of the target window;

  • the permitted edit radius and ambiguity policy have been chosen deliberately;

  • sample identifiers and FASTQ paths have been checked against the run sheet;

  • the intended downstream consumer of the output is known. For CRISPR counts, DotMatch writes a MAGeCK-style matrix but does not perform screen statistics.

Create a reviewable project from the release package:

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

cd crispr_evaluation
dotmatch assay check assay.toml

assay new samples the input reads to propose an extraction window. Review inference_report.json, the target-library audit, and the generated assay.toml. Keep status = "draft" until a qualified reviewer has confirmed the configuration, then set it to ready.

Run and review

./run.sh
dotmatch assay handoff assay.toml

./run.sh uses dotmatch assay start: it runs preflight, assignment, target audit, QC, and validation. Open these files in order:

  1. assay_out/reliability_report.html

  2. assay_out/sample_qc.tsv and assay_out/crispr_qc.html for CRISPR runs

  3. assay_out/assay_report.html

  4. assay_out/counts.mageck.tsv or the primary count/demultiplexing output

  5. assay_out/methods.md and assay_out/CITATION.bib

Do not treat a passed DotMatch reliability verdict as proof of biological validity. It means the configured target safety and software QC rules passed. Review positive/negative controls, sample identity, sequencing-run metrics, replicate agreement, and downstream analysis according to the local assay protocol.

Handoff package

dotmatch assay handoff assay.toml writes assay_out/handoff/ without copying raw reads. The bundle is suitable for an internal technical review or a workflow-maintainer evaluation:

  • README_FOR_REVIEW.md states the review order and boundary;

  • handoff_manifest.json records the configuration, verdict, input file sizes, and input/output SHA-256 hashes;

  • SHA256SUMS verifies the copied review files;

  • review/ contains reports, QC tables, primary outputs, methods, citation, and software-version records.

In the controlled workspace containing the FASTQs, recompute each input hash in handoff_manifest.json before approving the handoff. Do not send raw reads or identifiers outside the approved data-handling route merely to make a DotMatch review bundle.

Decision record

For each evaluated assay, record:

Item

Record

Assay and target-library revision

Name, source, checksum, and owner

Read extraction

Read, orientation, start, length, and rationale

Assignment rules

Metric, edit radius, ambiguity policy, and handling of ambiguous reads

Input identity

Sample-sheet revision and FASTQ checksums

QC outcome

Reliability verdict, findings reviewed, controls, and any exceptions

Output recipient

Count matrix/report location and downstream analysis owner

Software record

DotMatch version, native version, methods.md, and CITATION.bib

Keep this record with the project or laboratory notebook. It makes a later rerun auditable without claiming that DotMatch replaces the remainder of the assay or analysis workflow.