Amplicon Analyzer

Amplicon Analyzer is a static web app for amplicon FASTQ analysis. It runs read QC, paired-end overlap joining, reference alignment, mutation calling, and figure export from a browser page.

Open the app:

https://best916116-crypto.github.io/NGS_analyzer/

Features

• FASTQ, FASTQ.GZ, and joined FASTQ text input
• File or folder selection for numbered sample batches
• Automatic R1/R2 sample grouping and overlap-consensus joining
• Custom, Cas / CRISPR, TALE / TALEN, and Prime Editor target annotation
• Optional multi-site analysis
• Setting CSV import and template export
• QC table, mutation table, allele spectrum, selected-sample allele reports, summary JSON, and heatmap export
• Zoomable heatmap with full-figure and visible-viewport PNG export

Basic Use

1. Open the app.
2. Enter the site name and full reference amplicon sequence.
3. Select the assay type and exact target positions.
4. Add FASTQ files or choose a folder.
5. Adjust QC and alignment settings if needed. Leave Read limit per sample blank, or enter 0, to process all reads.
6. Run the analysis.
7. Export tables, summary files, and figures.

Inputs

Input	Notes
.fastq, .fq	Plain FASTQ
.fastq.gz, .fq.gz	Gzip-compressed FASTQ
.fastjoin, .fastqjoin, .fqjoin, .join, .txt	Joined FASTQ-style text
Site name	Label used in figures, reports, and site-specific filenames
Reference amplicon	Full expected PCR amplicon sequence
Setting CSV	Optional run configuration

For raw paired-end files, filenames with common R1 / R2 markers are grouped by sample. R2 reads are reverse-complemented and joined to R1 when a high-confidence overlap is found. Unjoined pairs are reported in the QC export.

If joined FASTQ files and raw R1/R2 files are provided for the same sample, joined files are used and raw files are skipped for that sample.

Read limit per sample is optional. When it is blank or 0, every parsed read is processed. Use a numeric limit only for quick validation runs because downstream read counts and allele frequencies will be based on that subset.

Setting CSV

The app can export a setting template with one row per amplicon site:

site_name,reference_amplicon_sequence,target_positions,assay_type,spacer_sequence,pam_sequence,spacer_window,tale_left_sequence,tale_right_sequence,tale_spacer_sequence,tale_padding,prime_edited_amplicon_sequence,prime_intended_edits,prime_ignore_regions,expected_edit,sample_start,sample_end,expected_sample_count,preferred_input,notes

Blank fields are ignored. assay_type may be custom, cas, tale, or prime. If assay_type is blank, the app infers the assay from filled spacer, PAM, TALE binding-site, or Prime Editor fields. For TALE / TALEN rows, the matched spacer is annotated as a targetable region; exact target bases come from target_positions.

Prime Editor mode separates intended and unwanted edits. Define intended edits with a full prime_edited_amplicon_sequence or a compact prime_intended_edits list such as C45T,52insA,del61. Use prime_ignore_regions for PBS, RTT, primer, or other regions that should remain in raw tables but be excluded from unwanted-edit signal.

Outputs

Output	Contents
processed_mutation_table.csv	Position-level mutation counts, intended/unwanted edit counts, and edit rates
target_window_table.csv	Target-window subset of the mutation table, including Prime Editor intended/unwanted columns
substitution_matrix.csv	Substitution counts by reference and observed base
allele_spectrum_table.csv	Unique allele sequences and edit signatures
allele_report_<site>_<sample>.svg	Publication-style allele spectrum report for the selected sample
allele_spectrum_<site>_<sample>.csv	Allele spectrum CSV for the selected sample
summary.mutations.csv	Legacy-compatible C/G mutation outcome summary by sample
summary.nonX_per_pos.mutations.csv	Legacy-compatible per-position non-reference mutation counts
qc_read_counts.csv	Read count, filtering, alignment, and paired-end join summary
amplicon_run_summary.json	Run settings, site summaries, QC totals, and top edits
amplicon_mutation_heatmap.svg	Editable vector heatmap
amplicon_mutation_heatmap.png	Full heatmap PNG
amplicon_mutation_heatmap_visible.png	Current heatmap viewport PNG
amplicon_run_report.md	Markdown run report
amplicon_run_report.html	Self-contained HTML report

Validation

Run the public FASTQ benchmark:

node tools/amplicon-analyzer-benchmark.mjs benchmarks/public/crispresso2_base_editor/config.json

Run the paired-end truth test:

node tools/validate-paired-end.mjs

The paired-end test generates matching raw R1/R2 and joined FASTQ fixtures, then checks that both analysis paths report the same aligned read count, target edit position, and edit percentage.

Repository Layout

.
|-- index.html
|-- docs/
|   `-- user_guide.html
|-- benchmarks/
|   `-- public/
|       `-- crispresso2_base_editor/
|-- tools/
|   |-- amplicon-analyzer-benchmark.mjs
|   `-- validate-paired-end.mjs
`-- README.md