EmMAIL is a command line tool for emm-type classification of Streptococcus pyogenes based on the isolate's whole genome sequence.
The use of EmMAIL will follow the dependencies of the tools within its pipeline, namely blastn and ispcr.
EmMAIL needs blastn and ispcr. As is, there is no way to automatically install both automatically with setup.py.
User (for now) will need to manually install the tools themselves and set them on $PATH for EmMAIL to work (EmMAIL expected both tools to be on $PATH).
EmMAIL requires Python >= 3. It requires Numpy and Scipy, both included in the setup.py.
EmMAIL uses setup.py for easier installation. Clone the GitHub page for EmMAIL, and run setup.py over the command line on the directory where setup.py for EmMAIL is located. The command is:
$ python3 setup.py installYou will then be able to use EmMAIL by calling emmail on the command line.
EmMAIL has 2 branches of usage: direct BLAST, or isPcr followed by BLAST. Product of any of the two pipelines will go through EmMAIL's clusterer to derive the type of the isolate.
The basic usage of EmMAIL is in the form of:
emmail ... [blast/pcr] ...On [blast/pcr], you are required to choose between blast or pcr to choose which pipeline you want. The first ellipsis ... is for EmMAIL's arguments. The second ellipsis ... is for arguments within the tools used in EmMAIL; blastn for BLAST pathway, and ispcr and blastn for PCR pathway.
These arguments are required for both of the pipelines:
| Argument | Variable Type | Description |
|---|---|---|
| --query | FASTA | An assembled genome FASTA |
| --db | blast DB | A BLAST database file |
While the optional arguments are:
| Argument | Variable Type | Default | Description |
|---|---|---|---|
| -clust_distance | integer | 500 | Distance between clusters to use |
| -output_type | string | short | Output type format. Choose within "short", "verbose", or "visual" |
| -saveIntermediary | boolean | False | On mention, do not remove intermediary files between tools |
| -outFinal | tsv | stdout | File to stream final output |
Options in blastn that can be manually changed for both BLAST and PCR pipeline.
| Argument | Variable Type | Default | Description |
|---|---|---|---|
| -dust | string | no | Filter query sequence with DUST |
| -perc_identity | integer | 95 | Minimal percent identity of sequence |
| -culling_limit | integer | 5 | Total hits to return in a single position |
| -mismatch | integer | 4 | Threshold number of mismatch to allow in BLAST hit |
| -align_diff | integer | 5 | Threshold for difference between alignment length and subject length in BLAST hit |
| -gap | integer | 2 | Threshold number of gap to allow in BLAST hit |
Options in ispcr that can be manually changed for the PCR pipeline. Aside from the optionals, the PCR pipeline has an additional required argument.
| Argument | Variable Type | Description |
|---|---|---|
| --primer | tsv | A tsv file containing primer set in the format "PrimerSetName\tPrimer1Sequence\tPrimer2Sequence" |
| Argument | Variable Type | Default | Description |
|---|---|---|---|
| -minPerfect | integer | 15 | Minimum size of perfect match at 3' primer end |
| -minGood | integer | 15 | Minimum size where there must be 2 matches for each mismatch |
| -maxSize | integer | 4000 | Positive integer value for maximum product length |
| -savePCR | boolean | False | On mention, PCR output file will not be automatically removed |
Again, you can also manually change the options in blastn within the PCR pipeline.
emmail --query isolate1.fa --db emm.fasta blast
emmail --query *.fa --db emm.fasta pcr --primer emmPrimer.tsv
emmail --query *.fa --db blastDB/emm.fasta -saveIntermediary blast -culling_limit 10 -align_diff 10
emmail --query Run19Jun/*.fa --db emm.fasta -output_type visual pcr --primer emmPrimer.tsv -maxSize 2000 -mismatch 5EmMAIL has three different result formats: short, verbose, and visual.
EmMAIL by default produces the short five tab-separated values to the command line. You can call -output_type <option> to choose the other two result format.
The short result returns: Isolate name_Number of clusters_Predicted type_Possible imposters_Answer's EMM cluster
While the verbose result returns: Isolate name_Number of BLAST hits_Number of clusters_Predicted type_Position in assembly_Possible imposters_Imposters position in assembly_Answer's EMM cluster, where the positions are presented in <contig_number>:<position_in_contig>.
The visual result returns an ASCII map of the emm-types in the genome. Types in a single contig are separated with "-", each representing 500bp distance from each other. Types found in different contigs are separated with tab.
The types are presented with flags when something is not right, the possible flags for now being:
| Flag | Description | Additional Information |
|---|---|---|
| * | Suspect Imposter | Types acknowledged in the CDC database as possibly not emm |
| ~ | Imperfect score | Match score below 100% |
Example for all result format:
Isolate1 1 EMM65.0 E6
Isolate2 3 EMM4.0 EMM236.3*;EMM156.0* E1
Isolate3 2 EMM52.1 EMM134.2* D4
Isolate1 6 1 EMM65.0 5:82168 E6
Isolate2 8 3 EMM4.0 2:104111 EMM236.3*;EMM156.0* 2:102762;2:105504 E1
Isolate3 5 2 EMM52.1 14:10502 EMM134.2* 5:913 D4
Isolate1 EMM65.0
Isolate2 EMM156.0*--EMM4.0--EMM236.3*
Isolate3 EMM52.1 EMM134.2*
If you are not sure which pipeline to choose from, I recommend using blast first, and use pcr when you want to check if anything weird is happening in your blast result.
An example problem where this might be useful is when there are too much hits reported by EmMAIL.
An important thing to note is that not all emm-like can be caught in the conventional PCR typing. PCR pipeline here can be used to see which hits would be returned in the setting of a conventional typing. This is however not fail-proof, as in silico PCR fails when the two primers do not align in the same contig. Better assembly would resolve this problem.
Should you want to fill issues or contact me about anything regarding EmMAIL, you can reach me here or on my email: andre.sutanto.91@gmail.com.