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NGI-AS-tech_note-2024

Introduction

This repository contains supplementary files for NGI tech note "Tech Note - Adaptive Sampling: targeted Oxford Nanopore long-read sequencing" published in 2024.

BED files

There are two sets of BED files with targets. One in hg38 co-ordinate space (in bedfiles_hg38/) and another one that requires a custom reference genome (found in bedfiles_customref).

In brief, the way to recreate the custom reference files is to use BEDTools getfasta on hg38 to extract the ROIs to ROI.fasta (rename the fasta entries to "ROI"), then use bedtools maskfasta to mask the ROI positions to hg38_masked.fasta Then cat ROI.fasta hg38_masked.fasta > custom_hg38.fasta The reason for using this custom reference method is to make it easier to validate in real-time on the instrument that enrichment is occuring:

Coverage of ROI shown in relation to the rest of the genome of an ongoing run in MinKNOW

experiment_sample hg38_bed custom_bed
P29602_01pct day1_0.1pct_targets.bed day1_0.1pct.bed
P29602_025pct day1_0.25pct_targets.bed day1_0.25pct.bed
P29602_05pct day1_0.5pct_targets.bed day1_0.5pct.bed
P29602_1pct day1_1pct_targets.bed day1_1pct.bed
P29602_4pct day1_4pct_targets.bed day1_4pct.bed
P29602_8pct day1_8pct_targets.bed day1_8pct.bed
P29602_ROI_as_ref day1_ROI_as_ref_1pct_targets.bed day1_ROI_as_ref_1pct.bed
P29602_01pct_10ROIs 1.bed  -
P29602_01pct_80ROIs 2.bed -
P29602_day3_gapfill day3_nomod_targets.bed day3_gapfill.bed
 Reserved channel experiment day1_8pct_targets.bed day1_8pct.bed

Command-line scripts

This is a collection of bespoke one-liners for processing the data in the tech note

Split channels in the reserved channel experiment

# bioawk: https://github.com/lh3/bioawk
# Channels 1-1499 with adaptive sampling
bioawk -cfastx 'match($comment, /ch=[[:digit:]]+ /){if(substr($comment, RSTART+3, RLENGTH-3) < 1500){print "@"$name" "$comment"\n"$seq"\n+\n"$qual}}' passed.fastq.gz | gzip -c > AS.fastq.gz
# Channels 1500-3000 without AS
bioawk -cfastx 'match($comment, /ch=[[:digit:]]+ /){if(substr($comment, RSTART+3, RLENGTH-3) >= 1500){print "@"$name" "$comment"\n"$seq"\n+\n"$qual}}' passed.fastq.gz | gzip -c > noAS.fastq.gz

Count active channels per time interval (fig 5)

# Method: Bin reads (passed + failed) into start_time hour. Find and count every 
# unique channel per bin. For future reference: just use nanoplot for this.

bioawk -cfastx '{print $comment}' AS_failed_and_passed.fastq.gz | grep -o "ch=\w* start_time=2023-\w\w-.*+02:00" | cut -b 4- | sed 's/ start_time=/ \t/' | grep -o ".*2023-09-[0-9]*T[0-9]*" | awk '{print $2":00:00\t"$1}' | sort | uniq | cut -f 1 | uniq -c | awk '{print $2"\t"$1}' > AS_chpr.txt

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