CLAWS (CNAG's Long-read Assembly Workflow in Snakemake)

Snakemake Pipeline used for de novo genome assembly @CNAG. It has been developed for Snakemake v6.0.5.

It accepts Oxford Nanopore Technologies (ONT) reads, PacBio HFi reads, illumina paired-end data, illumina 10X data and Hi-C reads. It does the preprocessing of the reads, assembly, polishing, purge_dups, scaffodling and different evaluation steps. By default it will preprocess the reads, run Flye + Hypo + purge_dups + yahs and evaluate the resulting assemblies with BUSCO, MERQURY, Nseries and assembly_stats. It needs a config file and a spec file (json file with instructions on which resources should slurm use for each of the jobs). Both files are created by the script "create_config_assembly.py" that is located in the bin directory. To check all the options accepted by the script, do:

bin/create_config_assembly.py -h

Once the 2 config files are produced, the pipeline can be launched using snakemake like this:

snakemake --notemp -j 999 --snakefile assembly_pipeline.smk --configfile assembly.config --is --cluster-conf assembly.spec --use-conda --use-envmodules

If you are using an HPC cluster, please check how should you run snakemake to launch the jobs to the cluster.

Most of the tools used will be installed via conda using the environments of the "envs" directory after providing the "--use-conda" option to snakemake. However, a few tools cannot be installed via conda and will have to be available in your PATH, or as a module in the cluster. Those tools are:

• NextDenovo/2.5.0
• NextPolish/1.4.1

How to provide input data:

There are several ways of providing the reads.

1- ONT reads

1.1 Using the option --ont-dir {DIR} in create_config_assembly.py.

If you do so, it will look for all the files in the directory that end in '.fastq.gz' and will add the basenames to "ONT_wildcards". These wildcards will be processed by the pipeline that will:

• Concatenate all the files into a single file

• Run filtlong with the default or specified parameters.

• Use the resulting file for assembly, polishing and/or purging.

You can also specify the basenames of the files that you want to use with the --ont-list option. In this case, the pipeline will use the wildcards that you're providing instead of merging all the files in the directory.

1.2 Using the option --ont-reads {FILE} in create_config_assembly.py.

If you do so, it will consider that you already have all the reads in one file and will:

• Run filtlong with the default or specified parameters.

• Use the resulting file for assembly, polishing and/or purging.

1.3 Using the option --ont-filt {FILE}. It will use this file as the output from filtlong. Hence, it will skip the preprocessing steps and directly use it for assembly, polishing and/or purging.

2-Illumina 10X-linked data

2.1 Using the --raw-10X {DIR:list} option.

Dictionary with 10X raw read directories, it has to be the mkfastq dir. You must specify as well the sampleIDs from this run. Example: '{"mkfastq- dir":"sample1,sample2,sample3"}'...

It will take each basename in the list to get the fastqs from the corresponding directory and run longranger on each sample. Afterwards, it will build meryldbs for each "barcoded" file. Finally, it will concatenate all the meryldbs and "barcoded" files. Resulting "barcoded" file will be used for polishing.

2.2 Using the --processed-10X {DIR} parameter.

This directory can already be there or be produced by the pipeline as described in step 2.1. Once all the "barcoded" fastq files are there, meryldbs will be built for each "barcoded" file. Finally, it will concatenate all the meryldbs and "barcoded" files. Resulting "barcoded" file will be used for polishing.

2.3 Using the --10X option.

The argument to this is the path to the concatenated ".barcoded" file that needs to be used for polishing. If the pre-concatenated files are not given, meryldbs will be directly generated with this file, but it may run out of memory.

3- Illumina short-read data

3.1 Using the --illumina-dir {DIR} option, that will look for all the files in the directory that end in '.1.fastq.gz' and will add the basenames to "illumina_wildcards". These wildcards will be processed by the pipeline that will:

• Trim adaptors with Trimgalore

• Concatenate all the trimmed *.1.fastq.gz and the *2.fastq.gz in one file per pair.

• The resulting reads will be used for building meryldbs and polishing.

3.2 Using the --processed-illumina option. If the directory exists and contains files, the pipeline will look for all the files in the directory that end in '.1.fastq.gz' and will add the basenames to "illumina_wildcards". These wildcards will be processed by the pipeline that will:

• Concatenate all the trimmed *.1.fastq.gz and the *2.fastq.gz in one file per pair.

• The resulting reads will be used for building meryldbs and polishing.

3.3 Using the --pe1 {FILE} and --pe2 {FILE} options. That will consider that these are the paired files containing all the illumina reads ready to be used and will build meryldbs and polish with them.

4- Input assemblies

If you want to polish an already assembled assembly, you can give it to the pipeline by using the option --assembly-in ASSEMBLY_IN [ASSEMBLY_IN ...] Dictionary with assemblies that need to be polished but not assembled and directory where they should be polished. Example: '{"assembly1":"polishing_dir1"}' '{"assembly2"="polishing_dir2"}' ...

If you want to start the pipeline after polishing on an already existing assembly, you can give it to the pipeline by using the option --postpolish-assemblies POSTPOLISH_ASSEMBLIES [POSTPOLISH_ASSEMBLIES ...] Dictionary with assemblies for which postpolishing steps need to be run but that are not assembled and base step for the directory where the first postpolishing step should be run. Example: '{"assembly1":"s04.1_p03.1"}' '{"assembly2"="s04.2_p03.2"}' ...

To evaluate and produce the final pretext file on a curated assembly, use --curated-assemblies CURATED_ASSEMBLIES [CURATED_ASSEMBLIES ...] Dictionary with assemblies that have already been curated. Evaluations and read alignment will be perforder. Example: '{"assembly1":"s04.1_p03.1"}' '{"assembly2":"s04.2_p03.2"}' ...

Description of implemented rules

1- Preprocessing:

• Read concatenation:

zcat {input.fastqs} | pigz -p {threads} -c > {output.final_fastq}

• Longranger for 10X reads: it uses the Longranger version installed in the path specified in the configfile

longranger basic --id={params.sample} --sample={params.sample} --fastqs={input.mkfastq_dir} --localcores={threads}

• Trimgalore: By default it gives the --max_n 0 --gzip -q 20 --paired --retain_unpaired options, but it can be changed with the --trim-galore-opts argument.

trim_galore -j {threads} {params.opts} {input.read1} {input.read2}

• Filtlong: it uses the Filtlong version installed in the path specified in the configfile. By default it gives the min_length and min_mean_q parameters, but extra parameters can be added with the --filtlong-opts option.

filtlong --min_length {params.minlen} --min_mean_q {params.min_mean_q} {params.opts} {input.reads} | pigz -p {threads} -c > {output.outreads}

• Build meryldb: it uses the merqury conda environment specified in the configfile. It takes as argument the --mery-k value that needs to be estimated first for the genome size. It can run either on the illumina reads, the ont reads or both, default behaviour is both.

meryl k={params.kmer} count output {output.out_dir} {input.fastq}

• Concat meryldbs: with the merqury conda environment specified in the configfile

meryl union-sum output {output.meryl_all} {input.input_run}

• Align ONT (Minimap2): it aligns the reads using minimap2 and outputs the alignment either in bam or in paf.gz formats. It uses the minimap2 conda environment specified in the configfile

minimap2 -{params.align_opts} -t {threads} {input.genome} {input.reads}

• Align Illumina (BWA-MEM): it aligns the reads with BWA-mem and outputs a bam file

bwa mem -Y {params.options} -t {threads} {input.genome} {input.reads} | samtools view -Sb - | samtools sort -@ {threads} -o {output.mapping} -

2- Assembly

• Flye (default). It is run by default, if you don't want the pipeline to run it, you can give --no-flye option when creating the config. It uses the conda environment specified in the config. By default it is set to 2 polishing iterations and gives the genome-size estimate that has been given when creating the config. Extra options can be provided with the --flye-opts.

flye --{params.readtype} {input.reads} -o {params.outdir}out -t {threads} -i {params.pol_iterations} {params.other_flye_opts}

• Nextdenovo (if run-nextdenovo): It uses the cluster module specified in the config. If nextdenovo option is turned on, the create_config script will also create the nextdenovo config file. Check the create_config help to see which options can be modified on it.

nextDenovo {input.config}

3- Polishing

• Hypo (default): It is the polisher that the pipeline uses by default, it can be turned off specifying --no-hypo when creating the config. If selected, the reads will be aligned in previous rules and then hypo will be run, it requires illumina data. It uses the conda environment specified in the config.

hypo -r @short_reads.list.txt -d {input.genome} -b {input.sr_bam} -c {coverage} -s {params.genome_size} -B {input.lr_bam} -t {threads} -o {output.polished} -p {params.proc} {params.opts}

• Nextpolish ont (if turned on): to run nextpolish with ONT reads, specify --nextpolish-ont-rounds and the number of rounds you want to run of it.

"python /apps/NEXTPOLISH/1.3.1/lib/nextpolish2.py -g {input.genome} -p {threads} -l lgs.fofn -r {params.lrtype} > {output.polished}

• Nextpolish illumina (if turned on): to run nextpolish with ONT reads, specify --nextpolish-ill-rounds and the number of rounds you want to run of it.

"python /apps/NEXTPOLISH/1.3.1/lib/nextpolish1.py -g {input.genome} -p {threads} -s {input.bam} -t {params.task} > {output.polished}

4- Post-assembly

• Purge_dups (by default): select --no-purgedups if you don't want to run it. If no manual cutoffs are given, it'll run purgedups with automatic cutoffs and then will rerun it selecting the mean cutoff as 0.75*cov. It uses the version installed in the cluster module specified in the config.

5- Evaluations

• Merqury: It runs on each 'terminal' assembly. This is, the base assembly and the resulting assembly from each branch of the pipeline.

• Busco: It can be run only in the terminal assemblies or on all the assemblies produced by the pipeline. It uses the conda environment specified in the config as well as the parameters specified.

• Nseries: This is run during the finalize on all the assemblies that are evaluated. After it, that rule combines the statistics produced by all the evaluation rules.

Description of all options

 bin/create_config_assembly.py -h
usage: create_configuration_file [-h] [--configFile configFile] [--specFile specFile] [--ndconfFile ndconfFile] [--concat-cores concat_cores]
                                 [--genome-size genome_size] [--lr-type lr_type] [--basename base_name] [--species species] [--keep-intermediate]
                                 [--preprocess-lr-step PREPROCESS_ONT_STEP] [--preprocess-10X-step PREPROCESS_10X_STEP]
                                 [--preprocess-illumina-step PREPROCESS_ILLUMINA_STEP] [--preprocess-hic-step PREPROCESS_HIC_STEP]
                                 [--flye-step FLYE_STEP] [--no-flye] [--nextdenovo-step NEXTDENOVO_STEP] [--run-nextdenovo]
                                 [--nextpolish-cores nextpolish_cores] [--minimap2-cores minimap2_cores] [--bwa-cores bwa_cores]
                                 [--hypo-cores hypo_cores] [--pairtools-cores pairtools_cores] [--busco-cores busco_cores]
                                 [--nextpolish-ont-rounds nextpolish_ont_rounds] [--nextpolish-ill-rounds nextpolish_ill_rounds]
                                 [--hypo-rounds hypo_rounds] [--longranger-cores longranger_cores] [--longranger-path longranger_path]
                                 [--genomescope-opts genomescope_additional] [--no-purgedups] [--ploidy ploidy] [--run-tigmint] [--run-kraken2]
                                 [--no-yahs] [--scripts-dir SCRIPTS_DIR] [--ont-reads ONT_READS] [--ont-dir ONT_DIR] [--ont-filt ONT_FILTERED]
                                 [--pe1 PE1] [--pe2 PE2] [--processed-illumina PROCESSED_ILLUMINA] [--raw-10X RAW_10X [RAW_10X ...]]
                                 [--processed-10X PROCESSED_10X] [--10X R10X] [--illumina-dir ILLUMINA_DIR]
                                 [--assembly-in ASSEMBLY_IN [ASSEMBLY_IN ...]]
                                 [--postpolish-assemblies POSTPOLISH_ASSEMBLIES [POSTPOLISH_ASSEMBLIES ...]]
                                 [--curated-assemblies CURATED_ASSEMBLIES [CURATED_ASSEMBLIES ...]] [--hic-dir HIC_DIR]
                                 [--pipeline-workdir PIPELINE_WORKDIR] [--filtlong-dir FILTLONG_DIR] [--concat-hic-dir CONCAT_HIC_DIR]
                                 [--flye-dir FLYE_DIR] [--nextdenovo-dir NEXTDENOVO_DIR] [--flye-polishing-dir POLISH_FLYE_DIR]
                                 [--nextdenovo-polishing-dir POLISH_NEXTDENOVO_DIR] [--eval-dir eval_dir] [--stats-out stats_out]
                                 [--hic-qc-dir hic_qc_dir] [--filtlong-minlen filtlong_minlen] [--filtlong-min-mean-q filtlong_min_mean_q]
                                 [--filtlong-opts filtlong_opts] [--kraken2-db kraken2_db] [--kraken2-kmer kraken2_kmers]
                                 [--kraken2-opts additional_kraken2_opts] [--kraken2-cores kraken2_threads] [--trim-galore-opts trim_galore_opts]
                                 [--trim-Illumina-cores Trim_Illumina_cores] [--flye-cores flye_cores] [--flye-polishing-iterations flye_pol_it]
                                 [--other-flye-opts other_flye_opts] [--nextdenovo-cores nextdenovo_cores] [--nextdenovo-jobtype nextdenovo_type]
                                 [--nextdenovo-task nextdenovo_task] [--nextdenovo-rewrite nextdenovo_rewrite]
                                 [--nextdenovo-parallel_jobs nextdenovo_parallel_jobs] [--nextdenovo-minreadlen nextdenovo_minreadlen]
                                 [--nextdenovo-seeddepth nextdenovo_seeddepth] [--nextdenovo-seedcutoff nextdenovo_seedcutoff]
                                 [--nextdenovo-blocksize nextdenovo_blocksize] [--nextdenovo-pa-correction  nextdenovo_pa_correction]
                                 [--nextdenovo-minimap_raw nextdenovo_minimap_raw] [--nextdenovo-minimap_cns nextdenovo_minimap_cns]
                                 [--nextdenovo-minimap_map nextdenovo_minimap_map] [--nextdenovo-sort nextdenovo_sort]
                                 [--nextdenovo-correction_opts nextdenovo_correction_opts] [--nextdenovo-nextgraph_opt nextdenovo_nextgraph_opt]
                                 [--sr-cov ill_cov] [--hypo-proc hypo_processes] [--hypo-no-lr] [--hypo-opts hypo_opts]
                                 [--purgedups-cores purgedups_cores] [--purgedups-calcuts-opts calcuts_opts] [--tigmint-cores tigmint_cores]
                                 [--tigmint-opts tigmint_opts] [--hic-qc] [--no-pretext] [--assembly-qc assembly_qc] [--yahs-cores yahs_cores]
                                 [--yahs-mq yahs_mq] [--yahs-opts yahs_opts] [--hic-map-opts hic_map_opts] [--mq mq [mq ...]]
                                 [--hic-qc-assemblylen hic_qc_assemblylen] [--blast-cores blast_cores] [--hic-blastdb blastdb]
                                 [--hic-readsblast hic_readsblast] [--no-final-evals] [--busco-lin busco_lineage] [--merqury-db merqury_db]
                                 [--merqury-plot-opts merqury_plot_opts] [--meryl-k meryl_k] [--meryl-threads meryl_threads]
                                 [--meryl-reads meryl_reads [meryl_reads ...]] [--ont-list ONT_wildcards] [--illumina-list illumina_wildcards]
                                 [--r10X-list r10X_wildcards] [--hic-list hic_wildcards]

Create a configuration json file for the assembly pipeline.

options:
  -h, --help            show this help message and exit

General Parameters:
  --configFile configFile
                        Configuration JSON to be generated. Default assembly.config
  --specFile specFile   Cluster specifications JSON fileto be generated. Default assembly.spec
  --ndconfFile ndconfFile
                        Name pf the nextdenovo config file. Default nextdenovo.config
  --concat-cores concat_cores
                        Number of threads to concatenate reads and to run filtlong. Default 4
  --genome-size genome_size
                        Approximate genome size. Example: 615m or 2.6g. Default None
  --lr-type lr_type     Type of long reads (options are flye read-type options). Default nano-hq
  --basename base_name  Base name for the project. Default None
  --species species     Name of the species to be assembled. Default None
  --keep-intermediate   Set this to True if you do not want intermediate files to be removed. Default False
  --preprocess-lr-step PREPROCESS_ONT_STEP
                        Step for preprocessing long-reads. Default 02.1
  --preprocess-10X-step PREPROCESS_10X_STEP
                        Step for preprocessing 10X reads. Default 02.2
  --preprocess-illumina-step PREPROCESS_ILLUMINA_STEP
                        Step for preprocessing illumina reads. Default 02.2
  --preprocess-hic-step PREPROCESS_HIC_STEP
                        Step for preprocessing hic reads. Default 02.3
  --flye-step FLYE_STEP
                        Step for running flye. Default 03.1
  --no-flye             Give this option if you do not want to run Flye.
  --nextdenovo-step NEXTDENOVO_STEP
                        Step for running nextdenovo. Default 03.2
  --run-nextdenovo      Give this option if you do want to run Nextdenovo.
  --nextpolish-cores nextpolish_cores
                        Number of threads to run the nextpolish step. Default 24
  --minimap2-cores minimap2_cores
                        Number of threads to run the alignment with minimap2. Default 32
  --bwa-cores bwa_cores
                        Number of threads to run the alignments with BWA-Mem2. Default 16
  --hypo-cores hypo_cores
                        Number of threads to run the hypo step. Default 24
  --pairtools-cores pairtools_cores
                        Number of threads to run the pairtools step. Default 100
  --busco-cores busco_cores
                        Number of threads to run BUSCO. Default 32
  --nextpolish-ont-rounds nextpolish_ont_rounds
                        Number of rounds to run the Nextpolish with ONT step. Default 0
  --nextpolish-ill-rounds nextpolish_ill_rounds
                        Number of rounds to run the Nextpolish with illumina step. Default 0
  --hypo-rounds hypo_rounds
                        Number of rounds to run the Hypostep. Default 1
  --longranger-cores longranger_cores
                        Number of threads to run longranger. Default 16
  --longranger-path longranger_path
                        Path to longranger executable. Default /scratch/project/devel/aateam/src/10X/longranger-2.2.2
  --genomescope-opts genomescope_additional
                        Additional options to run Genomescope2 with. Default -m 10000
  --no-purgedups        Give this option if you do not want to run Purgedups.
  --ploidy ploidy       Expected ploidy. Default 2
  --run-tigmint         Give this option if you want to run the scaffolding with 10X reads step.
  --run-kraken2         Give this option if you want to run Kraken2 on the input reads.
  --no-yahs             Give this option if you do not want to run yahs.

Inputs:
  --scripts-dir SCRIPTS_DIR
                        Directory with the different scripts for the pipeline. Default
                        /software/assembly/pipelines/Assembly_pipeline/CLAWSv2.2/bin/../scripts/
  --ont-reads ONT_READS
                        File with all the ONT reads. Default None
  --ont-dir ONT_DIR     Directory where the ONT fastqs are stored. Default None
  --ont-filt ONT_FILTERED
                        File with the ONT reads after running filtlong on them. Default None
  --pe1 PE1             File with the illumina paired-end fastqs, already trimmed, pair 1.
  --pe2 PE2             File with the illumina paired-end fastqs, already trimmed, pair 2.
  --processed-illumina PROCESSED_ILLUMINA
                        Directory to Processed illumina reads. Already there or to be produced by the pipeline.
  --raw-10X RAW_10X [RAW_10X ...]
                        Dictionary with 10X raw read directories, it has to be the mkfastq dir. You must specify as well the sampleIDs from this run.
                        Example: '{"mkfastq-dir":"sample1,sample2,sample3"}'...
  --processed-10X PROCESSED_10X
                        Directory to Processed 10X reads. Already there or to be produced by the pipeline.
  --10X R10X            File with barcoded 10X reads in fastq.gz format, concatenated.
  --illumina-dir ILLUMINA_DIR
                        Directory where the raw illumina fastqs are stored. Default None
  --assembly-in ASSEMBLY_IN [ASSEMBLY_IN ...]
                        Dictionary with assemblies that need to be polished but not assembled and directory where they should be polished. Example:
                        '{"assembly1":"polishing_dir1"}' '{"assembly2"="polishing_dir2"}' ...
  --postpolish-assemblies POSTPOLISH_ASSEMBLIES [POSTPOLISH_ASSEMBLIES ...]
                        Dictionary with assemblies for whic postpolishing steps need to be run but that are not assembled and base step for the
                        directory where the first postpolishing step should be run. Example: '{"assembly1":"s04.1_p03.1"}'
                        '{"assembly2":"s04.2_p03.2"}' ...
  --curated-assemblies CURATED_ASSEMBLIES [CURATED_ASSEMBLIES ...]
                        Dictionary with assemblies that have already been curated. Evaluations and read alignment will be perforder. Example:
                        '{"assembly1":"s04.1_p03.1"}' '{"assembly2":"s04.2_p03.2"}' ...
  --hic-dir HIC_DIR     Directory where the HiC fastqs are stored. Default None

Outputs:
  --pipeline-workdir PIPELINE_WORKDIR
                        Base directory for the pipeline run. Default /scratch_isilon/groups/assembly/jgomez/test_CLAWSv2/ilErePala/assembly/
  --filtlong-dir FILTLONG_DIR
                        Directory to process the ONT reads with filtlong. Default s02.1_p01.1_Filtlong
  --concat-hic-dir CONCAT_HIC_DIR
                        Directory to concatenate the HiC reads. Default s02.3_p01.1_Concat_HiC
  --flye-dir FLYE_DIR   Directory to run flye. Default s03.1_p02.1_flye/
  --nextdenovo-dir NEXTDENOVO_DIR
                        Directory to run nextdenovo. Default s03.2_p02.1_nextdenovo/
  --flye-polishing-dir POLISH_FLYE_DIR
                        Directory to polish the flye assembly. Default s04.1_p03.1_polishing/
  --nextdenovo-polishing-dir POLISH_NEXTDENOVO_DIR
                        Directory to run nextdenovo. Default s04.2_p03.2_polishing/
  --eval-dir eval_dir   Base directory for the evaluations. Default evaluations/
  --stats-out stats_out
                        Path to the file with the final statistics.
  --hic-qc-dir hic_qc_dir
                        Directory to run the hic_qc. Default hic_qc/

Filtlong:
  --filtlong-minlen filtlong_minlen
                        Minimum read length to use with Filtlong. Default 1000
  --filtlong-min-mean-q filtlong_min_mean_q
                        Minimum mean quality to use with Filtlong. Default 80
  --filtlong-opts filtlong_opts
                        Extra options to run Filtlong (eg. -t 4000000000)

Kraken2:
  --kraken2-db kraken2_db
                        Database to be used for running Kraken2. Default None
  --kraken2-kmer kraken2_kmers
                        Database to be used for running Kraken2. Default None
  --kraken2-opts additional_kraken2_opts
                        Optional parameters for the rule Kraken2. Default
  --kraken2-cores kraken2_threads
                        Number of threads to run the Kraken2 step. Default 16

Trim_Galore:
  --trim-galore-opts trim_galore_opts
                        Optional parameters for the rule trim_galore. Default --max_n 0 --gzip -q 20 --paired --retain_unpaired
  --trim-Illumina-cores Trim_Illumina_cores
                        Number of threads to run the Illumina trimming step. Default 8

Flye:
  --flye-cores flye_cores
                        Number of threads to run FLYE. Default 128
  --flye-polishing-iterations flye_pol_it
                        Number of polishing iterations to use with FLYE. Default 2
  --other-flye-opts other_flye_opts
                        Additional options to run Flye. Default --scaffold

Nextdenovo:
  --nextdenovo-cores nextdenovo_cores
                        Number of threads to run nextdenovo. Default 2
  --nextdenovo-jobtype nextdenovo_type
                        Job_type for nextdenovo. Default slurm
  --nextdenovo-task nextdenovo_task
                        Task need to run. Default all
  --nextdenovo-rewrite nextdenovo_rewrite
                        Overwrite existing directory. Default yes
  --nextdenovo-parallel_jobs nextdenovo_parallel_jobs
                        Number of tasks used to run in parallel. Default 50
  --nextdenovo-minreadlen nextdenovo_minreadlen
                        Filter reads with length < minreadlen. Default 1k
  --nextdenovo-seeddepth nextdenovo_seeddepth
                        Expected seed depth, used to calculate seed_cutoff, co-use with genome_size, you can try to set it 30-45 to get a better
                        assembly result. Default 45
  --nextdenovo-seedcutoff nextdenovo_seedcutoff
                        Minimum seed length, <=0 means calculate it automatically using bin/seq_stat. Default 0
  --nextdenovo-blocksize nextdenovo_blocksize
                        Block size for parallel running, split non-seed reads into small files, the maximum size of each file is blocksize. Default 1g
  --nextdenovo-pa-correction  nextdenovo_pa_correction
                        number of corrected tasks used to run in parallel, each corrected task requires ~TOTAL_INPUT_BASES/4 bytes of memory usage,
                        overwrite parallel_jobs only for this step. Default 100
  --nextdenovo-minimap_raw nextdenovo_minimap_raw
                        minimap2 options, used to find overlaps between raw reads, see minimap2-nd for details. Default -t 30
  --nextdenovo-minimap_cns nextdenovo_minimap_cns
                        minimap2 options, used to find overlaps between corrected reads. Default -t 30
  --nextdenovo-minimap_map nextdenovo_minimap_map
                        minimap2 options, used to map reads back to the assembly. Default -t 30 --no-kalloc
  --nextdenovo-sort nextdenovo_sort
                        sort options, see ovl_sort for details. Default -m 400g -t 20
  --nextdenovo-correction_opts nextdenovo_correction_opts
                        Correction options. Default -p 30 -dbuf
  --nextdenovo-nextgraph_opt nextdenovo_nextgraph_opt
                        nextgraph options, see nextgraph for details. Default -a 1

Hypo:
  --sr-cov ill_cov      Approximate short read coverage for hypo Default 0
  --hypo-proc hypo_processes
                        Number of contigs to be processed in parallel by HyPo. Default 6
  --hypo-no-lr          Set this to false if you don¡t want to run hypo with long reads. Default True
  --hypo-opts hypo_opts
                        Additional options to run Hypo. Default None

Purge_dups:
  --purgedups-cores purgedups_cores
                        Number of threads to run purgedups. Default 8
  --purgedups-calcuts-opts calcuts_opts
                        Adjusted values to run calcuts for purgedups. Default None

Scaffold_with_10X:
  --tigmint-cores tigmint_cores
                        Number of threads to run the 10X scaffolding step. Default 12
  --tigmint-opts tigmint_opts
                        Adjusted values to run the scaffolding with 10X reads. Default None

HiC:
  --hic-qc              Give this option if only QC of the HiC data needs to be done.
  --no-pretext          Give this option if you do not want to generate the pretext file
  --assembly-qc assembly_qc
                        Path to the assembly to be used perfom the QC of the HiC reads.
  --yahs-cores yahs_cores
                        Number of threads to run YAHS. Default 48
  --yahs-mq yahs_mq     Mapping quality to use when running yahs.Default 40
  --yahs-opts yahs_opts
                        Additional options to give to YAHS.Default
  --hic-map-opts hic_map_opts
                        Options to use with bwa mem when aligning the HiC reads. Deafault -5SP -T0
  --mq mq [mq ...]      Mapping qualities to use for processing the hic mappings. Default [0, 40]
  --hic-qc-assemblylen hic_qc_assemblylen
                        Lentgh of the assembly to be used for HiC QC
  --blast-cores blast_cores
                        Number of threads to run blast with the HiC unmapped reads.Default 8
  --hic-blastdb blastdb
                        BLAST Database to use to classify the hic unmapped reads. Default /scratch_isilon/groups/assembly/data/blastdbs
  --hic-readsblast hic_readsblast
                        Number of unmapped hic reads to classify with blast. Default 100

Finalize:
  --no-final-evals      If specified, do not run evaluations on final assemblies. Default True
  --busco-lin busco_lineage
                        Path to the lineage directory to run Busco with. Default None
  --merqury-db merqury_db
                        Meryl database. Default None
  --merqury-plot-opts merqury_plot_opts
                        Meryl database. Default None
  --meryl-k meryl_k     Merqury plot additional options, for example " -m 200 -n 6000|". Default None
  --meryl-threads meryl_threads
                        Number of threads to run meryl and merqury. Default 4
  --meryl-reads meryl_reads [meryl_reads ...]
                        Type of reads to be used to build the meryldb. Default ont illumina

Wildcards:
  --ont-list ONT_wildcards
                        List with basename of the ONT fastqs that will be used. Default None
  --illumina-list illumina_wildcards
                        List with basename of the illumina fastqs. Default None
  --r10X-list r10X_wildcards
                        List with basename of the raw 10X fastqs. Default None
  --hic-list hic_wildcards
                        List with basename of the raw hic fastqs. Default None

Changes made to v2.2:

1. General:

   Now default read_type is nano-hq

2. Rule trim_galore:

   "--max_n 0" has been added to the default behaviour of "--trim-galore-opts"

3. Meryl:

   New option "--meryl-reads" has been added to the config. Default is "Illumina ont" to build the meryl database using both type of reads, it can be changed to one or the other

4. Merqury:

   Option "--merqury-plot-opts" has been added to config file. It can be used to modify the x and y axis maximum values (eg. --merqury-plot-opts " -m 200 -n 6000")

5. Genomescope:

   "-m 10000" is now part of the default behavior of "--genomescope-opts"

6. Hic_statistics:

   This is now running for each assembly and mq for which a pretext file is generated

7. Assembly inputs for different steps:

   a. "--assembly-in" to start after assembly step (eg. Evaluation, polishing, purging and scaffolding)

   b. "--postpolish-assemblies" to start after polishing step (eg. Evaluation, purging and scaffolding)

   c. "--curated-assemblies" to start after scaffolding step (eg. Evaluation and pretext generation)