Use these commands to install the scripts
git clone https://github.com/LSHTMPathogenSeqLab/amplicon-seq.git
cd amplicon-seq
python setup.py install
First change directory to the repo on your computer and then run the following commands
cd /path/to/amplicon-seq
git pull
python setup.py install
usage: amplicon_script.py [-h] --index-file INDEX_FILE --read1 READ1 --read2
READ2 --ref REF --gff GFF --bed BED [--trim]
[--trim-qv TRIM_QV] [--min-base-qual MIN_BASE_QUAL]
Amplicon sequencing analysis script
optional arguments:
-h, --help show this help message and exit
--index-file INDEX_FILE
CSV file containing fields "Sample,I1,I2" (default:
None)
--read1 READ1 Forward read file (default: None)
--read2 READ2 Reverse read file (default: None)
--ref REF Reference fasta (default: None)
--gff GFF GFF file (default: None)
--bed BED BED file with genes/amplicon locations (default: None)
--trim Perform triming (default: False)
--trim-qv TRIM_QV Quality value to use in the sliding window analysis
(default: 20)
--min-base-qual MIN_BASE_QUAL
Minimum base quality to use by freebayes (default: 30)
Download the latest clinvar vcf to annotate variations with rsIDs and clinical significance (now required).
curl -s ftp://ftp.ncbi.nlm.nih.gov/pub/clinvar/vcf_GRCh38/clinvar.vcf.gz > clinvar_GRCh38.vcf.gz
tabix -f clinvar_GRCh38.vcf.gz
human_amplicon_script.py [-h] --index-file INDEX_FILE --read1 READ1 --read2 READ2
--ref REF --gff GFF --bed BED --clinVar CLINVAR
[--trim]
[--trim-qv TRIM_QV]
[--min-base-qual MIN_BASE_QUAL]
[--min-adf MIN_ADF]
[--vcf-files VCF_FILES [VCF_FILES ...]]
[--min-variant-qual MIN_VARIANT_QUAL]
[--min-sample-af MIN_SAMPLE_AF]
[--per-sample-only] [--version]
Amplicon sequencing analysis script
optional arguments:
-h, --help show this help message and exit
--index-file INDEX_FILE
CSV file containing fields "Sample,I1,I2" (default:
None)
--read1 READ1 Forward read file (default: None)
--read2 READ2 Reverse read file (default: None)
--ref REF Reference fasta (default: None)
--gff GFF GFF file (default: None)
--bed BED BED file with genes/amplicon locations (default: None)
--clinVar CLINVAR ClinVar SNP annotation file (default: None)
Position info file (default: None)
--trim Perform triming (default: False)
--trim-qv TRIM_QV Quality value to use in the sliding window analysis
(default: 20)
--min-base-qual MIN_BASE_QUAL
Minimum base quality to use by freebayes (default: 30)
--min-adf MIN_ADF Set a minimum frequency for a mixed call (default:
None)
--vcf-files VCF_FILES [VCF_FILES ...]
VCF files with positions to include (optional)
(default: None)
--min-variant-qual MIN_VARIANT_QUAL
Quality value to use in the sliding window analysis
(default: 30)
--min-sample-af MIN_SAMPLE_AF
Quality value to use in the sliding window analysis
(default: 0.05)
--per-sample-only Perform triming (default: False)
--version show program's version number and exit
Note: Run AFTER Demultiplexing by nanopore AND primer-specific barcodes
This step required for all species.
Place all demultiplexed fastq files into a directory and use these commands to generate a sample ID list in .csv format with 'sample' as the column header.
ls *.fastq | sed 's/.fastq//' > samples.txt
echo -e "sample" | cat - samples.txt > samples_header.txt
sed 's/ \+/,/g' samples_header.txt > sample_file.csv
This sample_file.csv will be used by both pipelines to perform mapping and variant calling for all samples within this file.
Finally, compress all FASTQ files prior to running either pipeline.
for f in *.fastq ; do bgzip -c "$f" > "${f%.*}.fastq.gz" ; done
General nanopore amplicon sequencing for use on multiple species (haploid and dipolid), such as Plasmodium and mosquito vectors.
Variants are annotated using bcftools consequence calling package.
nanopore_amplicon_script.py [-h] --index-file INDEX_FILE --ref REF --gff GFF --bed BED
[--min-base-qual MIN_BASE_QUAL]
[--version]
Nanopore amplicon sequencing analysis script
optional arguments:
-h, --help show this help message and exit
--index-file INDEX_FILE
sample_file.csv with the "sample" column for sample IDs (default: None)
--ref REF Reference fasta (default: None)
--gff GFF GFF file (default: None)
--bed BED BED file with genes/amplicon locations (default: None)
--min-base-qual MIN_BASE_QUAL
Minimum base quality to use by freebayes (default: 30)
--version show program's version number and exit
Human nanopore amplicon sequencing pipeline for use on human targets.
Variants annotated using the ClinVar database providing rsID and clinical significance annotations.
Requires ClinVar VCF to run.
curl -s ftp://ftp.ncbi.nlm.nih.gov/pub/clinvar/vcf_GRCh38/clinvar.vcf.gz > clinvar_GRCh38.vcf.gz
tabix -f clinvar_GRCh38.vcf.gz
human_nanopore_amplicon_script.py [-h] --index-file INDEX_FILE --ref REF --gff GFF --bed BED --clinVar CLINVAR
[--min-base-qual MIN_BASE_QUAL]
[--version]
Nanopore amplicon sequencing analysis script
optional arguments:
-h, --help show this help message and exit
--index-file INDEX_FILE
sample_file.csv with the "sample" column for sample IDs (default: None)
--ref REF Reference fasta (default: None)
--gff GFF GFF file (default: None)
--bed BED BED file with genes/amplicon locations (default: None)
--clinVar CLINVAR ClinVar SNP annotation file (default: None)
--min-base-qual MIN_BASE_QUAL
Minimum base quality to use by freebayes (default: 30)
--version show program's version number and exit
Create box plots using the plot_read_depth_human.r or plot_read_depth_Pfalciparum.r in the plots folder to show the read depth at positions of interest using the depth_info.txt output file from the amplicon sequencing pipeline.
Example using loci in humans associated with severe malarial disease
