Examples running GABI

Below are some examples of how you could approach the assembly of your genomes. We will assume that you use a site-specific config file named my_profile. See our installation guide for ways to configure the pipeline.

Illumina short-reads

This is probably the most common approach used for obtaining genomic information from bacterial isolates. Typically, you will have one set of paired-end reads per sample - so your samplesheet would look as follows:

sample  platform    fq1 fq2
SampleA ILLUMINA    /path/to/sampleA_R1.fastq.gz    /path/to/sampleA_R2.fastq.gz
SampleB ILLUMINA    /path/to/sampleB_R1.fastq.gz    /path/to/sampleB_R2.fastq.gz

With this samplesheet, GABI can then be run like so:

All default optionsUsing UnicyclerAssembly-only

Run GABI with all-defaults options.

nextflow run bio-raum/gabi -profile my_profile \
-r 1.4.0 \
--input samples.tsv \
--run_name MyIlluminaRun

Switch from Shovill to Unicycler for assembly.

nextflow run bio-raum/gabi -profile my_profile \
-r 1.4.0 \
--input samples.tsv \
--unicycler \
--run_name MyIlluminaRun 

Only assemble genomes and do not perform downstream characterization.

nextflow run bio-raum/gabi -profile my_profile \
-r 1.4.0 \
--input samples.tsv \
--skip_optional \
--run_name MyIlluminaRun 

ONT reads

Inexpensive long-reads generated using Nanopore sequencing are an attractive alternative to Illumina sequencing as the superior read length typically enables the reconstruction of the entire bacterial chromosome into one contig. Downsides include platform-specific homopolymer artifacts and a lower per-base quality, the latter of which can be largely compensated by added sequencing depth.

Nanopore data per sample is typically split across many read files - which you could either merge before running GABI or provide individually with the same sample ID. With Nanopore being a single-end technology, the fq2 remains empty, of course.

sample  platform    fq1 fq2
SampleA NANOPORE    /path/to/BBJ413_pass_barcode01_34ed0b48_e8967f4e_0.fastq.gz
SampleA NANOPORE    /path/to/BBJ413_pass_barcode01_34ed0b48_e8967f4e_1.fastq.gz
SampleA NANOPORE    /path/to/BBJ413_pass_barcode01_34ed0b48_e8967f4e_2.fastq.gz
SampleA NANOPORE    /path/to/BBJ413_pass_barcode01_34ed0b48_e8967f4e_3.fastq.gz
SampleB NANOPORE    /path/to/BBJ413_pass_barcode02_34ed0b48_e8967f4e_0.fastq.gz
SampleB NANOPORE    /path/to/BBJ413_pass_barcode02_34ed0b48_e8967f4e_1.fastq.gz
SampleB NANOPORE    /path/to/BBJ413_pass_barcode02_34ed0b48_e8967f4e_2.fastq.gz
SampleB NANOPORE    /path/to/BBJ413_pass_barcode02_34ed0b48_e8967f4e_3.fastq.gz

With this samplesheet, you can run GABI:

All default optionsHigh quality readsConsensus assemblyConsensus assembly with homopolish

Run GABI with all-defaults options.

nextflow run bio-raum/gabi -profile my_profile \
-r 1.4.0 \
--input samples.tsv \
--run_name MyOntRun

Run GABI on data generated with SUP basecalling

nextflow run bio-raum/gabi -profile my_profile \
-r 1.4.0 \
--input samples.tsv \
--onthq \
--run_name MyOntRun

Perform consensus assembly with Autocycler.

nextflow run bio-raum/gabi -profile my_profile \
-r 1.4.0 \
--input samples.tsv \
--autocycler \
--run_name MyOntRun

Perform consensus assembly with Autocycler and polish with Homopolish.

nextflow run bio-raum/gabi -profile my_profile \
-r 1.4.0 \
--input samples.tsv \
--autocycler \
--homopolish \
--run_name MyOntRun

ONT and Illumina reads

Combining Nanopore long reads with Illumina short reads will extend the assembly process by using short reads for assembly polishing. No additional command-line flags are required. A samplesheet with mixed ONT and Illumina reads will look as follows:

sample  platform    fq1 fq2
SampleA ILLUMINA    /path/to/sampleA_R1.fastq.gz    /path/to/sampleA_R2.fastq.gz
SampleA NANOPORE    /path/to/BBJ413_pass_barcode01_34ed0b48_e8967f4e_0.fastq.gz
SampleA NANOPORE    /path/to/BBJ413_pass_barcode01_34ed0b48_e8967f4e_1.fastq.gz
SampleA NANOPORE    /path/to/BBJ413_pass_barcode01_34ed0b48_e8967f4e_2.fastq.gz
SampleA NANOPORE    /path/to/BBJ413_pass_barcode01_34ed0b48_e8967f4e_3.fastq.gz
SampleB ILLUMINA    /path/to/sampleB_R1.fastq.gz    /path/to/sampleB_R2.fastq.gz
SampleB NANOPORE    /path/to/BBJ413_pass_barcode02_34ed0b48_e8967f4e_0.fastq.gz
SampleB NANOPORE    /path/to/BBJ413_pass_barcode02_34ed0b48_e8967f4e_1.fastq.gz
SampleB NANOPORE    /path/to/BBJ413_pass_barcode02_34ed0b48_e8967f4e_2.fastq.gz
SampleB NANOPORE    /path/to/BBJ413_pass_barcode02_34ed0b48_e8967f4e_3.fastq.gz

Note that Homopolish will not run if short reads are provided.

Pacbio reads

Assembling genomes form Pacbio reads is largely equivalent to using ONT reads, with the exception that Pacbio distinguishes between corrected (HiFi) and uncorrected (CLR) reads - which GABI needs to be told about. GABI only accepts one kind (HiFI or CLR) of Pacbio reads per run.

sample  platform    fq1 fq2
sampleA PACBIO  /path/to/sampleA_hifi.fastq.gz
sampleB PACBIO  /path/to/sampleB_hifi.fastq.gz

GABI can then be run like so:

CLR reads, default optionsHiFi reads, default optionsCLR reads with Homopolish

Run GABI with all-defaults options for CLR reads.

nextflow run bio-raum/gabi -profile my_profile \
-r 1.4.0 \
--input samples.tsv \
--run_name MyIlluminaRun

Run GABI with all-defaults options for HiFi reads.

nextflow run bio-raum/gabi -profile my_profile \
-r 1.4.0 \
--input samples.tsv \
--pacbio_hifi \
--run_name MyIlluminaRun

Run GABI with CLR reads and use Homopolish

nextflow run bio-raum/gabi -profile my_profile \
-r 1.4.0 \
--input samples.tsv \
--homopolish \
--run_name MyIlluminaRun

Pacbio and Illumina reads

Combining Pacbio long reads with Illumina short reads will extend the assembly process by using short reads for assembly polishing. No additional command-line flags are required. A samplesheet with mixed Pacbio and Illumina reads will look as follows:

sample  platform    fq1 fq2
SampleA ILLUMINA    /path/to/sampleA_R1.fastq.gz    /path/to/sampleA_R2.fastq.gz
sampleA PACBIO  /path/to/sampleA_hifi.fastq.gz
SampleB ILLUMINA    /path/to/sampleB_R1.fastq.gz    /path/to/sampleB_R2.fastq.gz
sampleB PACBIO  /path/to/sampleB_hifi.fastq.gz

Pre-assembled genomes

GABI also accepts pre-assembled genomes - in which case only limited QC data can be generated of course.

A samplesheet for pre-assembled genomes looks as follows:

sample  assembly
sampleA /path/to/sampleA.fasta
sampleB /path/to/sampleB.fasta

Then you can run GABI as usual:

All default settingsSkipping AMR predictions

nextflow run bio-raum/gabi -profile my_profile \
-r 1.4.0 \
--input samples.tsv \
--run_name MyAssemblies

Skip the optional AMR prediction steps

nextflow run bio-raum/gabi -profile my_profile \
-r 1.4.0 \
--input samples.tsv \
--skip_amr \
--run_name MyAssemblies