# DRAGEN Microbial Enrichment Plus ## Description DRAGEN Microbial Enrichment Plus (DME+), formerly known as the Explify Analysis Pipeline, offers a dedicated informatics solution with flexible analysis options for the following Illumina Infectious Disease and Microbiology target-capture enrichment panel kits: the Illumina Respiratory Pathogen ID/AMR Enrichment Panel Kit (RPIP), Illumina Urinary Pathogen ID/AMR Enrichment Panel Kit (UPIP), and Illumina Viral Surveillance Panel V2 Kit (VSP V2). The application delivers easy-to-use, powerful secondary analysis of Illumina sequencing data, with workflows for sample QC, viral WGS (whole-genome sequencing), pathogen detection and quantification, and antimicrobial resistance (AMR) marker profiling. It also supports custom reference sequence analysis. * RPIP: Target-capture enrichment of >280 RNA and DNA respiratory pathogens, including SARS-CoV-2, Influenza viruses, Respiratory syncytial virus, Mycobacterium and Legionella species, and >4000 AMR markers. * UPIP: Target-capture enrichment of >170 genitourinary pathogens, including fastidious, slow-growing, and anaerobic uropathogens, sexually transmitted microorganisms, and >4000 bacterial AMR markers. * VSP V2: Target-capture enrichment for whole-genome sequencing (WGS) of 200 RNA and DNA viruses prioritized as high-risk to public health, zoonotic surveillance, and biotech, and >200 viral AMR markers. * Custom: Analyze FASTQ/FASTA read files with a custom reference sequence database. Note that samples enriched using the Illumina Respiratory Virus Oligo Panel/Respiratory Virus Enrichment Kit (RVOP/RVEK) and Viral Surveillance Panel Kit (VSP) can also be analyzed using DME+ and the VSP V2 database. ## Pipeline Steps The following table describes the different steps performed by the pipeline, which steps apply to each panel, and whether the step is run when using a set of custom references. | Step | Description | Panels | Custom References | | :---------------------------: | :--------------------------------------------------------------------------------------------------------------------------------------------------------------------: | :----------: | :---------------: | | Read QC | Can be disabled. Low-quality bases are trimmed. Short and low-quality reads are discarded. It is assumed that appropriate adapter trimming has already been performed. | All | Yes | | Post-QC FASTQ Generation | Can choose to create a FASTQ with the trimmed reads, or a set of kingdom-specific FASTQs with the trimmed reads. Disabled by default. | All | Yes | | Dehosting | Removes human reads. | All | Yes | | Sample QC | Sample composition analysis and enrichment factor calculation (which requires an internal control). | All | No | | Microorganism Classification | K-mer-based analysis with configurable sensitivity. | VSP V2 | No | | Microorganism Detection | Alignment-based analysis and consensus generation. | All | Yes | | Microorganism Quantification | Requires an internal control. | All | No | | Bacterial AMR Marker Analysis | Nucleotide and protein alignment, consensus generation, variant calling and annotation. | RPIP, UPIP | No | | Viral Variant Calling | Detects variants from alignment results. | RPIP, VSP V2 | No | | Viral AMR Marker Analysis | Variant calling and annotation. | RPIP, VSP V2 | No | | Report Generation | Creates the AP JSON. | All | Yes | ## Command Line Settings | Option | Description | | ------------------------------------------- | ----------------------------------------------------------------------------------------------------------------------------------- | | Required Inputs | | | `--enable-explify` | Enables the DME+ pipeline. (Default=false). | | `--output-file-prefix` | Prefix for all output files. | | `--output-directory` | Directory for all output files. | | `--explify-sample-list` | Input sample list .tsv file with sample IDs, FASTQs, etc. | | `--explify-test-panel-name` | "RPIP", "UPIP", "VSP V2", "Custom". | | `--explify-test-panel-version` | Set to test panel version (e.g. "1.0.0"). | | `--explify-ref-db-dir` | Path to root directory for database files. | | Optional Inputs | | | `--intermediate-results-dir` | Area for temporary files. Size must be greater than size of all FASTQ files multiplied by 3. | | `--explify-load-db-ram` | Option to load database into RAM if not on ramdisk. (Default=false). | | `--explify-no-read-qc` | Option to turn off read QC on FASTQs before analysis. (Default=false). | | `--explify-internal-control` | Option to set internal control from an accepted list. (Default="Enterobacteria phage T7"). | | `--explify-internal-control-concentration` | Option to set internal control concentration. (Default=12100000). | | `--explify-ncpus` | Option to set the number of CPUs available for processing. | | `--explify-sensitivity-threshold` | Option to set sensitivity threshold for considering a virus present. Range: 0 < Integer < 1000. Only valid for VSP V2. (Default=5). | | `--explify-custom-ref-fasta` | Reference FASTA file. Required for Custom reference DBs. | | `--explify-custom-ref-bed` | Reference BED file. Optional for Custom reference DBs. | | `--explify-viral-consensus-depth-threshold` | Minimum depth at position to include base in viral consensus sequence. Only relevant for RPIP and VSP V2 (Default=1). | | `--explify-viral-vc-depth-threshold` | Minimum total depth at position to report viral variant. Only relevant for RPIP and VSP V2. (Default=5). | | `--explify-viral-vc-af-threshold` | Minimum allele frequency to report viral variant. Only relevant for RPIP and VSP V2. (Default=0.2). | | `--explify-post-qc-fastq-mode` | Create a single post-quality fastq file or files split by kingdom. Choices='off', 'single', 'split'. (Default=off). | ### Example Command Line ```shell dragen \ --enable-explify=true \ --output-file-prefix \ --explify-sample-list /path/to/sample/list/tsv \ --explify-test-panel-name <"RPIP"/"UPIP"/"VSP V2"/"Custom"> \ --explify-test-panel-version \ --explify-ref-db-dir /path/to/root/db/dir \ --explify-load-db-ram=true \ --output-directory \ --intermediate-results-dir \ --explify-ncpus=20 ``` ## Input Details ### Sample Input List Applies to: `--explify-sample-list` The sample input list is a column-formatted file with *tab* separations between the columns (i.e., a `.tsv` file). ``` SampleID BatchID RunID ControlFlag FastQs MySample MyBatch MyRun POS /path/to/fastq1.gz /path/to/fastq2.gz ``` Notes: * The **SampleID** values *must* be unique. * **BatchID** and **RunID** are to help users track and manage sample analyses. Often the **BatchID** is used to track libraries that were prepared together, and the **RunID** is used to track sequencing runs. They can also be left blank. * The **ControlFlag** value can be *POS*, *NEG*, *BLANK*, or left empty. * *POS* is used to indicate a positive control sample. * *NEG* is used to indicate a negative control sample. * *BLANK* is used to indicate a blank control sample (e.g. buffer only). * If there are multiple FASTQ files, they are tab delimited. * Please be very careful when editing tsv files. Some editors replace tabs with spaces without alerting the user. ### Internal Control Applies to: `--explify-internal-control`, `--explify-internal-control-concentration` The user may specify one of the internal controls listed below. If `NONE` is specified, the internal control concentration is ignored. These are case-sensitive and must be input exactly as they appear: * `Allobacillus halotolerans` * `Armored RNA Quant Internal Process Control` * `Enterobacteria phage T7` (This is the default) * `Escherichia virus MS2` * `Escherichia virus Qbeta` * `Escherichia virus T4` * `Imtechella halotolerans` * `Phocid alphaherpesvirus 1` * `Phocine morbillivirus` * `Truepera radiovictrix` * `NONE` The internal control concentration is an integer representing the number of *copies/mL of sample* for the internal control.