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<h1 id="pathgen-faq-main">NCBI Pathogen Detection Frequently Asked Questions (FAQ)</h1>
<h2 aria-expanded="true" id="where-can-i-find-more-informatio">Where can I find more information on the NCBI Pathogen Detection System</h2>
<p>Our <a href="/pathogens/pathogens_help/">help page</a> provides detailed documentation on how to use the NCBI Pathogen Detection System, including all of the ways to view the processed data in our browsers. Additional information on the organization of the FTP site and on analysis methods can be found on our FTP site in the <a href="https://ftp.ncbi.nlm.nih.gov/pathogen/ReadMe.txt">ReadMe.txt</a> and <a href="https://ftp.ncbi.nlm.nih.gov/pathogen/Methods.txt">Methods.txt</a> files.</p>
<h2 aria-expanded="false" id="what-is-the-pathogen-detection-p">What is the Pathogen Detection project?</h2>
<p><a href="/pathogens/">NCBI Pathogen Detection</a> project is a centralized system that
integrates sequence data and analysis for bacterial pathogens.</p>
<p>NCBI Pathogen Detection integrates bacterial and fungal pathogen genomic
sequences from numerous ongoing surveillance and research efforts whose sources
include food, environmental sources such as water or production facilities, and
patient samples. Foodborne, hospital-acquired, and other potentially clinically
infectious pathogens are included.</p>
<p>The system provides two major automated real-time analyses:</p>
<ol>
<li>It quickly clusters related pathogen genome sequences to identify potential
transmission chains, helping public health scientists investigate disease
outbreaks</li>
<li>As part of the National Database of Antibiotic Resistant Organisms (NDARO),
NCBI screens genomic sequences using AMRFinderPlus to identify the
antimicrobial resistance, stress response, and virulence genes found in
bacterial genomic sequences, which enables scientists to track the spread of
resistance genes and to understand the relationships among antimicrobial
resistance, stress response, and virulence.</li>
</ol>
<p>A number of public health agencies and researchers in the US and
internationally are collecting samples from clinical cases, from the
environment, from food products, and from industrial production facilities to
facilitate active, real-time surveillance of pathogens, including foodborne
disease. Public health agencies and researchers sequence the samples and submit
the data to NCBI, which analyzes the sequences and compares them to others in
its database, including all genomes in GenBank, to identify closely related
sequences. The aim is to identify closely or clonally related isolates to aid
in outbreak investigation. </p>
<p>The NCBI Pathogen Detection Project also analyzes the assemblies in its database in real-time for known anti-microbial resistance (AMR) genes and other genes of interest and maintains <a href="/pathogens/antimicrobial-resistance/">software and databases</a> to facilitate monitoring and research including the <a href="/pathogens/antimicrobial-resistance/">National Database of Antibiotic Resistant Organisms</a>.</p>
<h2 aria-expanded="false" id="what-is-a-targetisolate">What is a target/isolate?</h2>
<p>A target is an assembled genome for one pathogen isolate. Assemblies for this may not yet be available in GenBank.</p>
<h2 aria-expanded="false" id="what-are-these-accession-numbers">What are these Accession Numbers? What is a PDG?</h2>
<p>The Pathogen Detection system is currently using three series of Accession
Numbers. </p>
<ul>
<li>PDT# - isolate accession for each pathogen genome</li>
<li>PDG# - taxgroup accessions for each organism group</li>
<li>PDS# - SNP cluster accessions - are sets of isolates that have been determined by wgMLST or single-linkage clustering to be closely related for the purposes of facilitating outbreak and traceback investigations. Note - not all isolates within the taxgroups will be genetically similar enough to another isolate to fall into a SNP cluster. In other words there will be isolates in the taxgroup (PDG#) that will not have a PDS number assigned because they were not found sufficiently close to other isolates in our system to be clustered together.</li>
</ul>
<p>These Accession Numbers are distinct from those associated with nucleotide records in GenBank or RefSeq. These Accession Numbers are available in the <a href="/pathogens/isolates">Pathogen Detection Isolates Browser</a>, <a href="/pathogens/microbigge">MicroBIGG-E</a>, and in <a href="https://ftp.ncbi.nlm.nih.gov/pathogen/Results/">FTP</a> files.</p>
<p>Each of the accessions ends in a .version e.g., PDG000000004.355. A larger
version number is always more recent than a smaller version number.</p>
<p>Note that unlike many other NCBI resources the Pathogen Detection accessions
are not archival (the data they refer to is not guaranteed to persist
indefinitely) because of the rapid real-time reanalysis that happens in the
Pathogen Detection System. For that reason we recommend you download the
metadata associated with your analyses and save it locally. Check our <a href="/core/assets/pathogens/files/HowTo/how_to_cite_pathogen_detection.pdf">HowTo
Cite the Pathogen Detection Resource and the Data Contained
Within</a>
PDF for info on how to get the metadata for web-based analyses.</p>
<h2 aria-expanded="false" id="does-the-pathogen-detection-syst">Does the Pathogen Detection system use all genomes?</h2>
<p>For each organism group the system incorporates ALL assembled genomes submitted
to GenBank that are not flagged as anomalous in the <a href="/assembly">Assembly database</a>, and select sequences submitted to <a href="/sra">SRA</a> by public health agencies and
others. If you would like sequences you have submitted to SRA from the <a href="/pathogens/organisms/">supported organisms</a> to be included in the NCBI Pathogen Detection analysis please contact us at <a href="mailto:pd-help@ncbi.nlm.nih.gov">pd-help@ncbi.nlm.nih.gov</a>.</p>
<h2 aria-expanded="false" id="what-about-other-types-of-bacter">What about other types of bacterial pathogens?</h2>
<p>NCBI Pathogen Detection now analyzes over <a href="/pathogens/organisms/">50 taxa</a>, and
more are addded as capacity allows. If you are interested in large-scale
real-time analyses of pathogens that are not included in the <a href="/pathogens/organisms/">Organism
Groups we analyze</a>, please contact us at
<a href="mailto:pd-help@ncbi.nlm.nih.gov">pd-help@ncbi.nlm.nih.gov</a>.</p>
<h2 aria-expanded="false" id="where-are-the-analysis-results-h">Where are the analysis results? How can I visualize the results?</h2>
<p>Results can be viewed in the <a href="/pathogens/isolates">Isolates Browser</a>,
<a href="/pathogens/microbigge">MicroBIGG-E</a>, and by
<a href="https://ftp.ncbi.nlm.nih.gov/pathogen/Results">FTP</a> approximately every
day if new data are submitted. Our system outputs SNP cluster trees in
*.pdf, *.newick, and *.asn format. The *.asn format can be opened
in <a href="/tools/gbench/">NCBI's Genome Workbench tool</a>. The phylogenetic
trees for the SNP clusters are available on both
<a href="https://ftp.ncbi.nlm.nih.gov/pathogen/Results/">FTP</a> and in the
<a href="/pathogens/isolates">isolates browser</a>.
<a href="/pathogens/antimicrobial-resistance/AMRFinder/">AMRFinderPlus</a> results
are available in the Microbial Browser for the Identification of Genetic and Genomic Elements (<a href="/pathogens/microbigge">MicroBIGG-E</a>).</p>
<p>More information can be found in our <a href="/pathogens/pathogens_help/">help documentation</a> and <a href="https://ftp.ncbi.nlm.nih.gov/pathogen/ReadMe.txt">FTP ReadMe file</a>.</p>
<h2 aria-expanded="false" id="how-are-isolates-clustered-and-w">How are isolates clustered and what are SNP clusters?</h2>
<!--
All isolates for a given taxonomic group are compared to each other, either by
a shared K-mer approach for smaller groups or by wgMLST for those groups with
sufficient numbers of assemblies to develop a good wgMLST scheme.
For taxonomic groups with wgMLST schemes all isolates that have fewer than 25
different alleles at shared loci are grouped into a "SNP cluster" for SNP-based
analysis and phylogenetic tree inference.
Taxonomic groups that are grouped by shared Kmers are clustered first into
larger clusters by Kmer analysis then within each of those "Kmer clusters" all
assemblies are aligned to each-other and those assemblies within 50 filtered
SNPs of each-other are grouped into a "SNP cluster" for SNP-based phylogenetic
tree inference.
-->
<p>See the <a href="/pathogens/pathogens_help/#data-processing-overview">Pipeline overview</a>
on our help page for a high level description of the pipeline including clustering, and the
<a href="https://ftp.ncbi.nlm.nih.gov/pathogen/Methods.txt">Methods.txt</a> on our FTP
site for more details.</p>
<h2 aria-expanded="false" id="how-are-snp-distances-computed">How are SNP distances computed?</h2>
<p>SNP distances are patristic distances on the maximum compatibility tree. See
below and the (<a href="/pubmed/28231758">Cherry, J. 2017. A practical exact maximum compatibility
algorithm for reconstruction of recent evolutionary history. BMC Bioinformatics:
2017 Feb 23;18(1):127. doi: 10.1186/s12859-017-1520-4</a>)
for more information on SNP distance calculation. For a description of
filtering of potential SNP sites that occurs before running compat see the
<a href="https://ftp.ncbi.nlm.nih.gov/pathogen/Methods.txt">Methods.txt</a>
file on FTP.</p>
<h2 aria-expanded="false" id="i-know-there-are-some-snps-separ">I know there are some SNPs separating these two isolates but in your cluster you are showing them 0 SNPs apart, why?</h2>
<p>We have several filtering steps that may be filtering out the SNPs that you see. In order to handle varying quality and error levels of sequence and assembly SNP filtering is required. See our <a href="https://ftp.ncbi.nlm.nih.gov/pathogen/Methods.txt">Methods.txt</a> file for information on SNP filtering. Individual SNPs and filtering is indicated in the VCF file provided for each of the SNP clusters.</p>
<h2 aria-expanded="false" id="why-doesnt-your-cluster-correspo">Why doesn't your cluster correspond to this published cluster? What are you doing different?</h2>
<p>Our clusters are based solely on automated analysis of isolate sequences. Published outbreaks generally consider other, epidemiological factors, as they should. </p>
<h2 aria-expanded="false" id="can-i-add-my-own-isolates-to-the">Can I add my own isolates to the cluster and recompute it?</h2>
<p>If you submit your sequence and let us know it will be included in the full
analysis pipeline. See our <a href="/pathogens/submit-data/">How to submit</a> page for
more information on how to submit your data.</p>
<p>However, the Pathogen Detection system is a high-throughput, automated system
and we are unable to customize SNP cluster contents, so if the pipeline does
not identify your isolate as being sufficiently close to others in a cluster it
will not be included in a cluster.</p>
<h2 aria-expanded="false" id="i-know-these-isolates-are-near-t">I know these isolates are near to the outbreak isolates, but they don't appear in the cluster</h2>
<p>See the above answer about how isolates are clustered. There are some possible data issues that may cause isolates that are in fact closely related to appear too distantly related. While we have extensive filtering steps to minimize the chances of this happen it can sometimes occur when the sequence data is contaminated or poor-quality.</p>
<h2 aria-expanded="false" id="i-know-these-isolates-are-more-t">I know these isolates are more than 50 SNPs away from the outbreak isolates, but is there any way I can get these included in the cluster to see exactly what the SNP distance is?</h2>
<p>No. The Pathogen Detection system is a high-throughput, automated system and we are unable to customize SNP cluster contents.</p>
<h2 aria-expanded="false" id="what-is-the-snp-threshold-for-an">What is the SNP threshold for an outbreak?</h2>
<p>The number of SNPs that define an oubreak is variable and depends on many factors. Outbreak determinations should be made in concert with epidemiological factors, and the number and identity of SNPs that identify an outbreak are dependent on these factors.</p>
<h2 aria-expanded="false" id="how-do-i-know-when-new-analysis-">How do I know when new analysis results are released?</h2>
<p>The pipeline produces new analysis results every day if new data has arrived for each organism group. The <a href="/pathogens/">Pathogen Detect home page</a> shows the most recent results with Accession Numbers, count of newly added and total isolates, and a link to each taxonomic group subset in the Isolates Browser.</p>
<p>The <a href="https://ftp.ncbi.nlm.nih.gov/pathogen/Results/">FTP</a> directory contains links to the latest kmer analysis and SNP analysis for each organism.</p>
<p>Automatic emails can notify you in real-time when there are updates for any Isolates Browser search. For more information see our Isolates Browser help for <a href="/pathogens/pathogens_help/#automated-searches">Automatic E-mail Notifications of New Data</a>.</p>
<h2 aria-expanded="false" id="how-do-i-submit-data">How do I submit data?</h2>
<p>Information on how to submit can be found <a href="/pathogens/submit-data/">here</a>.</p>
<h2 aria-expanded="false" id="how-do-i-submit-betalactamase-mc">How do I submit beta-lactamase, MCR, or Qnr alleles?</h2>
<p>Information on how to submit for allele assignment can be found <a href="/pathogens/submit-beta-lactamase/">here</a></p>
<h2 aria-expanded="false" id="how-do-i-submit-and-find-ast-dat">How do I submit and find AST data?</h2>
<p>To <a href="/pathogens/submit-data/#ast">submit AST data</a>.</p>
<p>To <a href="/pathogens/isolates/#AST_phenotypes:*">find all isolates with BioSample records in the isolates browser</a>.</p>
<p>To <a href="/biosample/?term=antibiogram%5bfilter%5d">find all BioSample records that have AST data</a>.</p>
<h2 aria-expanded="false" id="how-do-i-contact-the-pathogen-de">How do I contact the Pathogen Detection team?</h2>
<p>The Pathogen Detection team can be reached by email at <a href="mailto:pd-help@ncbi.nlm.nih.gov">pd-help@ncbi.nlm.nih.gov</a>.</p>
<h2 aria-expanded="false" id="what-are-the-amr_genotypes-and-a">What are the AMR_genotypes and AST_phenotypes column?</h2>
<p>These two columns refer to antimicrobial resistance.</p>
<p>The AST_phenotypes column corresponds to the antibiogram submission into BioSample database that captures antibiotic susceptbility phenotypic testing (MICs or disk diffusion). Only isolates that have an antibiogram submitted will show AST data. See the help document for the <a href="/pathogens/pathogens_help/#isolates-browser-data-field-ASTPhenotypes">AST phenotypes column</a>.</p>
<p>The AMR_genotypes column corresponds to antimicrobial resistance genes that
have been identified by NCBI's
<a href="/pathogens/antimicrobial-resistance/AMRFinder/">AMRFinderPlus</a>. This process
is run on all isolates submitted by collaborators contributing data for the
Pathogen Detection system, and is also run against all
GenBank genomes that are incorporated into the <a href="/pathogens/isolates">Isolates
Browser</a> (although there may
be a delay in the analysis). See also the help document on the <a href="/pathogens/pathogens_help/#isolates-browser-data-field-AMRGenotypes">AMR genotypes
column</a>.
For more info about NCBI's efforts about antimicrobial resistance gene/protein
identification, see <a href="/pathogens/antimicrobial-resistance/">Antimicrobial
Resistance</a>.
For information on the software we developed to identify AMR genes see
<a href="/pathogens/antimicrobial-resistance/AMRFinder/">AMRFinderPlus</a>. For
information on the reference set of genes and proteins see the <a href="/pathogens/refgene/">Reference Gene
Catalog</a>.</p>
<h2 aria-expanded="false" id="how-are-the-phylogenetic-trees-r">How are the phylogenetic trees reconstructed?</h2>
<p>The SNP trees are calculated using a maximum compatibility algorithm that is efficient for closely related bacteria while dealing with input data quality issues. The algorithm is described in this paper (<a href="/pubmed/28231758">Cherry, J. 2017. A practical exact maximum compatibility algorithm for reconstruction of recent evolutionary history. BMC Bioinformatics: 2017 Feb 23;18(1):127. doi: 10.1186/s12859-017-1520-4</a>). The VCF file output has a field that lists incompatible sites that were filtered by compat.</p>
<h2 aria-expanded="false" id="software_list">Can I download the software and run it myself?</h2>
<p>Unfortunately the complete current pipeline is tied very tightly to many NCBI internal resources and so it can't be run outside of our environment. We would like to make a distributable version, but we have not yet had development capacity to do so. Several pieces of software devloped by NCBI and used in the pipeline are available including:</p>
<ul>
<li>AMR, stress resistance, and virulence gene identification software AMRFinderPlus <a href="https://pubmed.ncbi.nlm.nih.gov/31427293/">Paper</a> and <a href="https://github.com/ncbi/amr/wiki">Software</a></li>
<li>The assemblers SKESA and SAUTE <a href="https://pubmed.ncbi.nlm.nih.gov/30286803/">Paper</a> and <a href="https://github.com/ncbi/skesa">Software</a></li>
<li>The maximum compatibility phylogenetic inference software "compat" <a href="https://pubmed.ncbi.nlm.nih.gov/28231758/">Paper</a> and <a href="https://ftp.ncbi.nih.gov/pub/jcherry/compat/">Software</a></li>
<li>The PGAP annotation system <a href="https://pubmed.ncbi.nlm.nih.gov/29112715/">Paper</a> and <a href="https://github.com/ncbi/pgap">Software</a></li>
</ul>
<h2 aria-expanded="false" id="is-your-snp-pipeline-published">Is your SNP pipeline published?</h2>
<p>Not yet. We're working on it :-)</p>
<p>In the meantime several of the components NCBI developed for the pipeline are published and open source <a href="#software_list">See above</a> and <a href="/pathogens/pathogens_help/#references">pipeline references</a>).</p>
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