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The objective of this tutorial is to illustrate the complete workflow of a chewBBACA pipeline for creating a wgMLST and a cgMLST schema for a colection of 714 Streptococcus agalactiae genomes (32 complete genomes and 682 draft genome assemblies deposited on the NCBI databases) by providing step-by-step instructions and displaying the obtained outputs.

All information about the NCBI genomes used in this example is on the .tsv file inside the genomes folder.

Please start by going through the following steps:

1. Install chewBBACA. Check Installing chewBBACA for instructions on how to install chewBBACA. chewBBACA includes Prodigal training files for several species, including for Streptococcus agalactiae. You can check the list of available training files here. We have included the training file for Streptococcus agalactiae in this repository.
2. Clone this repository to the local folder of your choice. To clone, run the following command:
   git clone https://github.com/B-UMMI/chewBBACA_tutorial
3. Go to the top-level directory of the cloned repository, .../chewBBACA_tutorial/, and run unzip genomes/complete_genomes.zip to extract all the complete genomes.

The execution times reported in this tutorial were obtained for a DELL XPS13 (10th Generation Intel® Core™ i7-10710U Processor - 12MB Cache, up to 4.7 GHz, using 6 cores). Using a computer with less powerful specifications can greatly increase the duration of the analyses.

The commands used in this tutorial assume that the working directory is the top-level directory of the cloned repository, .../chewBBACA_tutorial/. The commands should be modified if they are executed from a different working directory. We have included the expected results for each section in the expected_results folder for reference (each subfolder has the name of one of the sections).

We will start by creating a wgMLST schema based on 32 Streptococcus agalactiae complete genomes (32 genomes with a level of assembly classified as complete genome or chromossome) available at NCBI. The sequences are present in the complete_genomes/ directory. To create the wgMLST schema, run the following command:

chewBBACA.py CreateSchema -i complete_genomes/ -o tutorial_schema --ptf Streptococcus_agalactiae.trn --cpu 6

The schema seed will be available at tutorial_schema/schema_seed. We passed the value 6 to the --cpu parameter to use 6 CPU cores, but you should pass a value based on the specifications of your machine. In our system, the process took 56 seconds to complete resulting on a wgMLST schema with 3128 loci. At this point the schema is defined as a set of loci each with a single representative allele.

The next step is to perform allele calling with the wgMLST schema created in the previous step for the 32 complete genomes. The allele call step determines the allelic profiles of the analyzed strains, identifying known and novel alleles in the analyzed genomes. Novel alleles are assigned an allele identifier and added to the schema. To perform allele call, run the following command:

chewBBACA.py AlleleCall -i complete_genomes/ -g tutorial_schema/schema_seed -o results32_wgMLST --cpu 6

The allele call used the default BSR threshold of 0.6 (more information on the threshold here) and took approximately 17 minutes to complete (an average of 32 seconds per genome). The allele call identified 14,720 novel alleles and added those alleles to the schema, increasing the number of alleles in the schema from 3,128 to 17,848.

The next step in the analysis is to determine if some of the loci can be considered paralogs, based on the result of the wgMLST allele calling. The Allele call returns a list of Paralogous genes in the RepeatedLoci.txt file that can be found on the results32_wgMLST/results_<datestamp> folder. The RepeatedLoci.txt file contains a set of 20 loci that were identified as possible paralogs. These loci should be removed from the schema due to the potential uncertainty in allele assignment (for a more detailed description see the Alelle Calling entry on the wiki). To remove the set of 20 paralogous loci from the allele calling results, run the following command:

chewBBACA.py RemoveGenes -i results32_wgMLST/results_<datestamp>/results_alleles.tsv -g results32_wgMLST/results_<datestamp>/RepeatedLoci.txt -o results32_wgMLST/results_<datestamp>/results_alleles_NoParalogs.tsv

This will remove the columns matching the 20 paralogous loci from the allele calling results and save the allelic profiles into the results_alleles_NoParalogs.tsv file (the new file contains allelic profiles with 3108 loci).

We can now determine the set of loci in the core genome based on the allele calling results. The set of loci in the core genome is determined based on a threshold of loci presence in the analysed genomes. We can run the TestGenomeQuality module to determine the impact of several threshold values on the number of loci in the core genome.

chewBBACA.py TestGenomeQuality -i results32_wgMLST/results_<datestamp>/results_alleles_NoParalogs.tsv -n 13 -t 200 -s 5 -o results32_wgMLST/results_<datestamp>/genome_quality_32

The process will automatically open a HTML file with the following plot:

larger image fig 1 or see interactive plot online

A set of 1136 loci were found to be present in all the analyzed complete genomes, while 1267 loci were present in at least 95%. For further analysis only the 1267 loci present in at least 95% of the complete genomes will be used. We selected that threshold value to account for loci that may not be identified due to sequencing coverage and assembly problems.

We can run the ExtraCgMLST module to quickly determine the set of loci in the core genome at 95%.

chewBBACA.py ExtractCgMLST -i results32_wgMLST/results_<datestamp>/results_alleles_NoParalogs.tsv -o results32_wgMLST/results_<datestamp>/cgMLST_95 --t 0.95

The list with the 1267 loci in the core genome at 95% is in the results32_wgMLST/results_<datestamp>/cgMLST_95/cgMLSTschema.txt file. This file can be passed to the --gl parameter of the AlleleCall process to perform allele calling only for the set of genes that constitute the core genome.

682 assemblies of Streptococcus agalactiae available on NCBI were downloaded (03-08-2016, downloadable zip file here, run unzip GBS_Aug2016.zip to extract genome files into a folder named GBS_Aug2016) and analyzed with MLST in order to exclude possibly mislabeled samples as Streptococcus agalactiae. Out of the 682 genomes, 2 (GCA_000323065.2_ASM32306v2 and GCA_001017915.1_ASM101791v1) were detected as being of a different species/contamination and were removed from the analysis.

Allele call was performed on the bona fide Streptococcus agalactiae 680 genomes using the 1267 loci that constitute the core genome at 95%. Paralog detection found no paralog loci.

chewBBACA.py AlleleCall -i path/to/GBS_Aug2016/ -g tutorial_schema/schema_seed --gl results32_wgMLST/results_<datestamp>/cgMLST_95/cgMLSTschema.txt -o results680_cgMLST --cpu 6

The process took approximately 39 minutes to complete (an average of 3.4 secs per genome).

We can now concatenate the cgMLST results for the 32 complete genomes with the cgMLST results for the 680 genomes to have all the results in a single file. To concatenate the allelic profiles of both analyses run the following command:

chewBBACA.py JoinProfiles -p1 results32_wgMLST/results_<datestamp>/cgMLST_95/cgMLST.tsv -p2 results680_cgMLST/results_<datestamp>/results_alleles.tsv -o cgMLST_all.tsv

The concatenated file was analyzed in order to assess the cgMLST allele quality attribution for all the genomes.

chewBBACA.py TestGenomeQuality -i cgMLST_all.tsv -n 13 -t 300 -s 5

larger image here fig 2 or see interactive plot online

While the number of loci present in 95% of genomes remains virtually constant at around 1200 loci, considering all or most of the genomes (90%<x≤100%) the number of loci present is lower and presents some variation when specific genomes are removed from the analysis.

We selected the results at the threshold of 25 for further analysis. Although this selection is somewhat arbitrary, when moving to a lower threshold there is step increase in the number of loci present in 95% and 99% of genomes that could represent the exclusion of a more divergent clade from the analysis. Furthermore, at the threshold of 25 there is an acceptable number of loci present in all considered genomes (650 genomes/440 loci), which we felt would afford a good discriminatory power.

The genomes that were removed at each threshold are indicated in the file analysis_all/removedGenomes.txt and a file analysis_all/removedGenomes_25.txt was created with only the genomes removed at the 25 threshold.

The following command creates a directory analysis_all/cgMLST_25/ and saves the cgMLST schema selected at the chosen threshold to the file cgMLST.tsv.

chewBBACA.py ExtractCgMLST -i cgMLST_all.tsv -o cgMLST_25 --g removedGenomes_25.txt

analysis_all/cgMLST_25/cgMLST.tsv was uploaded to Phyloviz online and can be accessed here

Since the quality of the used assemblies was not confirmed, it is possible that some of the assemblies included were of low quality. A general analysis of the assemblies show a N50 variation that ranges from 8055 to over 2.2M, while the number of contigs ranges between 1 and 553. These results made us suspect that the quality of the genomes could have affected the allele call results and consequently caused a significant drop in the number of loci detected as present in all genomes.

As stated previously, to obtain the cgMLST schema, some genomes (n=62) had to be removed since they were extremes cases of missing data. In order to assess the possible reason for their poor allele call performance, two plots were built. The removed genomes were then highlighted and dashed lines were drawn linking the values for the same genomes.

The first plot represents the total number of bp in contigs with a size >10 kbp and the N50 of the assemblies, sorted by decreasing values.

larger image fig 3

The second plot represents the total number of contigs and the number of contigs >10kbp.

larger image fig 4

See interactive plot online

At first sight, most of the removed genomes (56/62) were located on the lower range of N50 and bp in contigs >10 kbp (fig.3) and the higher number of contigs (fig.4).

The 5 genomes that were outside this pattern were individually checked:

1. GCA_000186445.1 here - 21 contigs but only 1 is above 10k (Scaffold with lot of Ns, 134 real contigs)
2. GCA_000221325.2 here- NCBI curated it out of RefSeq because it had a genome length too large
3. GCA_000427055.1 here- NCBI curated it out of RefSeq because it had many frameshifted proteins
4. GCA_000289455.1 here- No ST found. We concluded the assembly has a problem but we have not yet identified it.
5. GCA_000288835.1 here- NCBI curated it out of RefSeq because it had many frameshifted proteins

Schema Evaluator was run on the cgMLST schema:

chewBBACA.py SchemaEvaluator -i tutorial_schema/schema_seed/ -o schema_evaluation --cpu 6

See the schema evaluator page here