Nanopore sequencing of CRISPR-Cas9 edited Pieris brassicae NSP gene — 24 barcoded amplicon libraries on MinION
Revised Workflow →This project assesses CRISPR-Cas9 editing efficiency at the Nitrile Specifier Protein (NSP) gene in Pieris brassicae. A ~500 bp PCR amplicon spanning the sgRNA target site was sequenced on an Oxford Nanopore MinION (R10.4.1 flow cell). 24 barcoded samples were multiplexed in a single run. Reads are aligned to the genomic reference to characterize indel patterns at the CRISPR cut site.
The reference is a 699 bp region from P. brassicae chromosome 5
(NC_059669.1:14882048-14885601) containing the first coding exon of the NSP gene,
flanked by intronic/intergenic sequence.
| File | Description |
|---|---|
NSP_region_PCR_sgRNA_genomicDNA.fasta | Multi-entry fasta: genomic DNA, exon, primers, sgRNA (with gap-alignment) |
NSP_genomic_ref.fasta | Clean genomic reference (699 bp, gaps removed) — used for alignment |
NSP_genomic_ref.mmi | Minimap2 index |
R10.4.1 flow cell, HAC basecalling, demultiplexed to 24 barcodes
Per-barcode quality assessment of raw reads
Map ONT reads to 699 bp genomic reference
Sort & index with samtools for IGV viewing
Characterize CRISPR edits at sgRNA cut site
# Align a barcode to reference (ONT preset, output sorted BAM)
minimap2 -a -x map-ont --MD NSP_genomic_ref.mmi barcode01/*.fastq.gz | \
samtools sort -o barcode01_aligned.bam -
samtools index barcode01_aligned.bam
# FastQC
fastqc barcode01/*.fastq.gz -o . --threads 2| Barcode | Reads | Aligned | FastQC |
|---|---|---|---|
| barcode01 | 15,119 | Done | Done |
| barcode02 | 12,126 | Done | Pending |
| barcode03 | 11,008 | Done | Pending |
| barcode04 | 8,910 | Done | Pending |
| barcode05 | 6,940 | Done | Pending |
| barcode06 | 10,323 | Done | Pending |
| barcode07 | 17,108 | Done | Pending |
| barcode08 | 13,009 | Done | Pending |
| barcode09 | 27,921 | Done | Pending |
| barcode10 | 5,685 | Done | Pending |
| barcode11 | 21,097 | Done | Pending |
| barcode12 | — | Pending | Pending |
| barcode13 | — | Pending | Pending |
| barcode14 | — | Pending | Pending |
| barcode15 | — | Pending | Pending |
| barcode16 | — | Pending | Pending |
| barcode17 | — | Pending | Pending |
| barcode18 | — | Pending | Pending |
| barcode19 | — | Pending | Pending |
| barcode20 | — | Pending | Pending |
| barcode21 | — | Pending | Pending |
| barcode22 | — | Pending | Pending |
| barcode23 | — | Pending | Pending |
| barcode24 | — | Pending | Pending |
| Parameter | Value |
|---|---|
| Platform | Oxford Nanopore MinION |
| Flow cell | R10.4.1 (FBF81064) |
| Basecaller | dna_r10.4.1_e8.2_400bps_hac v5.2.0 |
| Run date | 2026-03-05 |
| Protocol | NSP_500bp_trial |
| Sample | NSP_Pnapi_500bp |
| Barcoding | 24 barcodes + unclassified |
| Target organism | Pieris brassicae (Large White butterfly) |
| Gene | NSP — Nitrile Specifier Protein (LOC123709534) |
| Reference | NC_059669.1:14882048-14885601 (chromosome 5) |
| Tool | Version | Purpose |
|---|---|---|
| minimap2 | 2.30 | ONT read alignment (-x map-ont) |
| samtools | 1.23 | BAM sorting, indexing, stats |
| FastQC | 0.12.1 | Read quality assessment |
| IGV | — | Visual inspection of alignments |