amplicon-coverage-inspector 1.15.250702

Visualization of coverage for amplicon sequencing

Amplicon Coverage Inspector (aci)

Amplicon Coverage Inspector (aci) is a bioinformatics tool designed to analyze the depth of amplicons using samtools. It provides a convenient way to determine the coverage and depth of specified regions in a BAM file based on a BED file using a user-generated bedfile.

Installation

pip install amplicon_coverage_inspector

From github

git clone https://github.com/erinyoung/ACI.git
cd ACI
pip install .

Dependencies

• python3.7+
  • pandas
  • matplotlib
  • pysam
  • intervaltree

Usage

aci --bam input.bam --bed amplicon.bed --out out

Final files are

• out/amplicon_depth.csv : csv file of the depth of each amplicon
• out/amplicon_depth.png : boxplot of information from csv file
• out/amplicon_depth_${sample}.bam.png : per-sample amplicon depth image with max and min
• out/amplicon_min_depth.csv : csv file for the minimum depth/unique depth of each amplicon
• out/amplicon_min_depth.png : boxplot of information from csv file
• out/overall_depth.csv : csv file of overall depth of bam file
• out/${reference}_depth.png : overal depth accross reference from overall_depth.csv

There are not currently options to change the look of the final image file. Instead, the amplicon_depth.csv file contains all the values in a '.csv' format that can be read into R, python, excel, or another tool for visualization.

Options

usage: aci [-h] -b BAM [BAM ...] -d BED [-o OUT] [-log LOGLEVEL] [-t THREADS] [-v]

options:
  -h, --help            show this help message and exit
  -b BAM [BAM ...], --bam BAM [BAM ...]
                        (required) input bam file(s)
  -d BED, --bed BED     (required) amplicon bedfile
  -o OUT, --out OUT     directory for results
  -log LOGLEVEL, --loglevel LOGLEVEL
                        logging level
  -t THREADS, --threads THREADS
                        specifies number of threads to use
  -v, --version         print version and exit

Bed file format

ACI is not very strict on the bed file format as only the first four columns are used.

The four columns (tab-delimited only) are

1. Reference (must the same as the reference of the bam file)
2. Start position
3. Stop position
4. Name of the amplicon

ACI does not support bedfiles with headers.

Example amplicon bedfile.

MN908947.3	54	385	1	1	+
MN908947.3	342	704	2	2	+
MN908947.3	664	1004	3	1	+
MN908947.3	965	1312	4	2	+
MN908947.3	1264	1623	5	1	+
MN908947.3	1595	1942	6	2	+
MN908947.3	1897	2242	7	1	+
MN908947.3	2205	2568	8	2	+
MN908947.3	2529	2880	9	1	+
MN908947.3	2850	3183	10	2	+

Amplicon bedfile

Primers should be trimmed out of the bam file prior to ACI regardless of whether the sequencing was paired or single-end. Primer sequences force portions of DNA to match reference and mask SNPs and other variants, so it is a normal request to trim primers out first.

Please be careful to not use a primer scheme bedfile because they are not the same. For an example, let's take a left and right primer based off of the reference MN908947.3. If left primer is expected to bind to 30-54 of MN908947.3, and the right primer is expected to bind to 385-410, the expected amplicon would be from 55-384.

ACI uses the user-provided amplicon bedfile to identify regions of the genome where both read1 and read2 are bounded by the start and stop location of that bedfile. If using a bam file generated with mapping single-end reads, only that one read must be within bounds. Any reads that map out of that specific region will be excluded. Then coverage is determined for that region. Therefore, all intended sequences are included. Nearby overlapping amplicons can still mask problematic primers/primer pairs and this should be taken into account when evaluating the output of ACI.

Testing

This repository contains a test bam and bed file in the /tests/data subdirectory.

aci -b tests/data/test.bam -d tests/data/test.bed -o testing

The resulting image should look something like the following.

Expected run time

file(s)	number of reads	threads	Elapsed (wall clock) time (h:mm:ss or m:ss)	Percent of CPU this job got	Maximum resident set size (kbytes)
SRR28446008.primertrim.bam	48777166	1	24:57.42	91%	65642916
SRR28446008.primertrim.bam	48777166	2	17:34.11	141%	41789232
SRR28446008.primertrim.bam	48777166	3	14:44.27	168%	35364164
SRR28446008.primertrim.bam	48777166	4	11:40.18	218%	24623868
SRR28446008.primertrim.bam	48777166	5	10:01.77	258%	20829232
SRR28446008.primertrim.bam	48777166	6	10:35.90	252%	22735132
SRR28446008.primertrim.bam	48777166	7	9:40.83	280%	16731812
SRR28446008.primertrim.bam	48777166	8	8:30.76	325%	16694240
SRR28446008.primertrim.bam	48777166	9	8:51.93	313%	17393304
SRR28446008.primertrim.bam	48777166	10	7:41.89	374%	12754412
SRR28446008.primertrim.bam	48777166	11	7:28.65	396%	13153432
SRR28446008.primertrim.bam	48777166	12	7:03.99	401%	13854976
SRR28446008.primertrim.bam	48777166	13	6:18.10	475%	11594264
SRR28446008.primertrim.bam	48777166	14	6:13.00	486%	12477736
SRR28446008.primertrim.bam	48777166	15	6:17.68	483%	13362504
SRR28446008.primertrim.bam	48777166	16	6:02.38	503%	14244760
SRR28446008.primertrim.bam, SRR28446040.primertrim.bam, SRR28446049.primertrim.bam, SRR28446053.primertrim.bam, SRR28446063.primertrim.bam, SRR28446123.primertrim.bam, SRR28446153.primertrim.bam	326156244	1	3:29:22	90%	94658408
SRR28446008.primertrim.bam, SRR28446040.primertrim.bam, SRR28446049.primertrim.bam, SRR28446053.primertrim.bam, SRR28446063.primertrim.bam, SRR28446123.primertrim.bam, SRR28446153.primertrim.bam	326156244	2	2:10:49	156%	58148124
SRR28446008.primertrim.bam, SRR28446040.primertrim.bam, SRR28446049.primertrim.bam, SRR28446053.primertrim.bam, SRR28446063.primertrim.bam, SRR28446123.primertrim.bam, SRR28446153.primertrim.bam	326156244	3	1:37:32	214%	56828032
SRR28446008.primertrim.bam, SRR28446040.primertrim.bam, SRR28446049.primertrim.bam, SRR28446053.primertrim.bam, SRR28446063.primertrim.bam, SRR28446123.primertrim.bam, SRR28446153.primertrim.bam	326156244	4	1:24:22	252%	56665120
SRR28446008.primertrim.bam, SRR28446040.primertrim.bam, SRR28446049.primertrim.bam, SRR28446053.primertrim.bam, SRR28446063.primertrim.bam, SRR28446123.primertrim.bam, SRR28446153.primertrim.bam	326156244	5	1:14:18	303%	57152076
SRR28446008.primertrim.bam, SRR28446040.primertrim.bam, SRR28446049.primertrim.bam, SRR28446053.primertrim.bam, SRR28446063.primertrim.bam, SRR28446123.primertrim.bam, SRR28446153.primertrim.bam	326156244	6	1:07:54	336%	57187576
SRR28446008.primertrim.bam, SRR28446040.primertrim.bam, SRR28446049.primertrim.bam, SRR28446053.primertrim.bam, SRR28446063.primertrim.bam, SRR28446123.primertrim.bam, SRR28446153.primertrim.bam	326156244	7	1:00:55	378%	57510668
SRR28446008.primertrim.bam, SRR28446040.primertrim.bam, SRR28446049.primertrim.bam, SRR28446053.primertrim.bam, SRR28446063.primertrim.bam, SRR28446123.primertrim.bam, SRR28446153.primertrim.bam	326156244	8	57:55.42	409%	57734304
SRR28446008.primertrim.bam, SRR28446040.primertrim.bam, SRR28446049.primertrim.bam, SRR28446053.primertrim.bam, SRR28446063.primertrim.bam, SRR28446123.primertrim.bam, SRR28446153.primertrim.bam	326156244	9	54:34.33	425%	65642916
SRR28446008.primertrim.bam, SRR28446040.primertrim.bam, SRR28446049.primertrim.bam, SRR28446053.primertrim.bam, SRR28446063.primertrim.bam, SRR28446123.primertrim.bam, SRR28446153.primertrim.bam	326156244	10	53:24.72	456%	57246404
SRR28446008.primertrim.bam, SRR28446040.primertrim.bam, SRR28446049.primertrim.bam, SRR28446053.primertrim.bam, SRR28446063.primertrim.bam, SRR28446123.primertrim.bam, SRR28446153.primertrim.bam	326156244	11	54:16.05	468%	58304164
SRR28446008.primertrim.bam, SRR28446040.primertrim.bam, SRR28446049.primertrim.bam, SRR28446053.primertrim.bam, SRR28446063.primertrim.bam, SRR28446123.primertrim.bam, SRR28446153.primertrim.bam	326156244	12	51:31.58	496%	58392632
SRR28446008.primertrim.bam, SRR28446040.primertrim.bam, SRR28446049.primertrim.bam, SRR28446053.primertrim.bam, SRR28446063.primertrim.bam, SRR28446123.primertrim.bam, SRR28446153.primertrim.bam	326156244	13	49:36.17	522%	58520260
SRR28446008.primertrim.bam, SRR28446040.primertrim.bam, SRR28446049.primertrim.bam, SRR28446053.primertrim.bam, SRR28446063.primertrim.bam, SRR28446123.primertrim.bam, SRR28446153.primertrim.bam	326156244	14	49:06.47	532%	59454204
SRR28446008.primertrim.bam, SRR28446040.primertrim.bam, SRR28446049.primertrim.bam, SRR28446053.primertrim.bam, SRR28446063.primertrim.bam, SRR28446123.primertrim.bam, SRR28446153.primertrim.bam	326156244	15	49:32.36	552%	59145092
SRR28446008.primertrim.bam, SRR28446040.primertrim.bam, SRR28446049.primertrim.bam, SRR28446053.primertrim.bam, SRR28446063.primertrim.bam, SRR28446123.primertrim.bam, SRR28446153.primertrim.bam	326156244	16	48:27.36	563%	344725513

Larger genomes, more coverage, and larger amplicons increase the computation time. Use -t or --threads to take advantage of determining the coverage of amplicons in parallel.

Contributing

Contributions are welcome! If you find any issues or have suggestions for improvements, please feel free to open an issue or submit a pull request.

Why this exists

I just needed an individual tool that evaluates how effective a set of primers are for an amplicon or bait-based NGS library prep. Similar scripts are included in many workflows including that of (artic)[https://github.com/artic-network/artic-ncov2019], but I needed something that was standalone. Samtools has a function, ampliconstats, that predicts amplicons based on a primer schema bedfile, but has errors when there are large number of primer pairs, the primer pairs overlap too much, are named outside of expected values, and can incorrectly pair primers when determining amplicons. This means that I needed control as to what was in the amplicon file.

License

This project is licensed under the MIT License.

Contact

For any questions or inquiries, please submit an issue.