The example pipelines are written in bash scripting language. The reference files used in both examples are included in the demo data. To run in haphpipe, execute one of the following lines:

haphpipe_assemble_01 read1.fq.gz read2.fq.gz ../refs/HIV_B.K03455.HXB2.fasta ../refs/HIV_B.K03455.HXB2.gtf sampleID

haphpipe_assemble_02 read1.fq.gz read2.fq.gz ../refs/HIV_B.K03455.HXB2.amplicons.fasta sampleID

Pipeline 1 implements amplicon assembly using a de novo approach. Reads are error-corrected and used to refine the initial assembly, with up to 5 refinement steps.

Pipeline 2 implements amplicon assembly using a reference-based mapping approach. Reads are error-corrected and used to refine the initial assembly, with up to 5 refinement steps.

Pipeline 1: haphpipe_assemble_01

This pipeline implements de novo assembly. Reads are first trimmed (trim_reads) and used as input for denovo assembly (assemble_denovo). The de novo assembly stage automatically performs error correction on the trimmed reads. The assembled contigs are used as input for amplicon assembly (assemble_amplicons) along with reference FASTA and GTF files. The assembly is then iteratively refined up to five times (refine_assembly) by mapping corrected reads to the assembled FASTA file and lastly finalized (finalize_assembly), resulting in a FASTA file with final consensus sequences, final VCF, and aligned BAM file.

To see the input information for Pipeline 1, use the -h option again like so: haphpipe_assemble_01 -h, and it will show the output:

USAGE:
haphpipe_assemble_01 [read1] [read2] [reference_fasta] [reference_gtf] [samp_id] <outdir>

----- HAPHPIPE assembly pipeline 01 -----

This pipeline implements amplicon assembly using a denovo approach. Reads are
error-corrected and used to refine the initial assembly, with up to 5
refinement steps.

Input:
read1:             Fastq file for read 1. May be compressed (.gz)
read2:             Fastq file for read 2. May be compressed (.gz)
reference_fasta:   Reference sequence (fasta)
reference_gtf:     Amplicon regions (GTF)
samp_id:           Sample ID
outdir:            Output directory (default is [sample_dir]/haphpipe_assemble_01)

To use this pipeline with your own data, replace [read1] with your read1 fastq file, [read2] with your read2 fastq file, [reference_fasta] with the reference fasta file of your choice, [reference_gtf] with the reference GTF file corresponding to the reference fasta file, and [samp_id] with the sample ID label. See examples below:

General command to execute pipeline 1:

haphpipe_assemble_01 samp/read1.fq.gz samp/read2.fq.gz refs/ref.fasta refs/ref.gtf samp

Example command to run with demo samples:

haphpipe_assemble_01 SRR8525886/SRR8525886_1.fastq SRR8525886/SRR8525886_2.fastq refs/HIV_B.K03455.HXB2.fasta refs/HIV_B.K03455.HXB2.gtf SRR8525886 SRR8525886

Pipeline 2: haphpipe_assemble_02

This pipeline implements reference-based mapping assembly. Reads are first trimmed (trim_reads) and error-corrected (ec_reads). The corrected reads are used as input for reference-based mapping assembly (refine_assembly) for up to five iterations. Lastly, the assembly is finalized (finalize_assembly) by mapping reads onto the refined reference sequence. The final output is a FASTA file with final consensus sequences, final VCF, and aligned BAM file.

To see the input information for Pipeline 2, use the -h option again like so: haphpipe_assemble_02 -h, and it will show the output:

USAGE:
haphpipe_assemble_02 [read1] [read2] [amplicons_fasta] [samp_id] <outdir>

----- HAPHPIPE assembly pipeline 02 -----

This pipeline implements amplicon assembly using a reference-based approach.
Reads are error-corrected and aligned to provided amplicon reference with up to
five refinement steps.

Input:
read1:             Fastq file for read 1. May be compressed (.gz)
read2:             Fastq file for read 2. May be compressed (.gz)
amplicons_fasta:   Amplicon reference sequence (fasta)
samp_id:           Sample ID
outdir:            Output directory (default is sample_dir/haphpipe_assemble_02)

To use this pipeline with your own data, replace [read1] with your read1 fastq file, [read2] with your read2 fastq file, [amplicons_fasta] with the reference fasta file containing as many amplicons as you desire, and [samp_id] with the sample ID label. See examples below:

General command to execute pipeline 2:

haphpipe_assemble_02 samp/read1.fq.gz samp/read2.fq.gz refs/ref.fasta samp

Example command to run with demo samples:

haphpipe_assemble_02 SRR8525886/SRR8525886_1.fastq SRR8525886/SRR8525886_2.fastq refs/HIV_B.K03455.HXB2.amplicons fasta SRR8525886 SRR8525886