Update on NCBI Pathogen Detection and Antimicrobial Resistance ActivitiesWilliam Klimke -GenomeTrakr, Sept 26-28, Crystal City, VA

Shared Network pathways and data streams for outbreak detection and investigations

Sampling of clinical/human, food, animal, and environmental bacterial isolates

Minimal metadata, everything publically accessible More extensive metadata, analyzed seq data (tools to translate)

data shared among PulseNet labs only

PulseNet National Database

CDC analysis (all raw sequence goes into NCBI

USDAFSIS labs

State health labs (clinical)

FSIS & FDA labsfood, environmental

PulseNetGenomeTrakrState agriculture & food regulatory labs

NCBI Submission Portal

Raw Genomic Sequence data

Analysis goals

1. Are these isolates clonally related?

2. What is the anti-microbial resistance gene repertoire of this isolate

NC

BI

Subm

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BioSamples

SRA

GenBank

BioProject

NCBI Pathogen Pipeline

Genome Assembly

Genome Annotation

Clustering

SNP analysis

Tree Construction

Reports

QC

USA

UK

Aus

Clin

ical

Pathogen Detection Pipeline

tpublished

NC

BI

Subm

issi

on P

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l

BioSamples

SRA

GenBank

BioProject

NCBI Pathogen Pipeline

Genome Assembly

Genome Annotation

Clustering

SNP analysis

Tree Construction

Reports

QC

USA

UK

Aus

Clin

ical

tsubmitted

G1

Neighbors

Goal: Improve Turnaround Time from Submission to Report

Pipeline Changes

• SKESA assembler releasedhttps://github.com/ncbi/SKESASKESA publication accepted (Genome Biology)

• SKESA running on all new submissions

• SKESA reassembly for all four foodborne pathogens

Pipeline Changes

• wgMLST schemes developed for all foodborne pathogens

• wgMLST now running for all four foodborne pathogens• used for rapid reports (within one hour of submission report

a table of nearest neighbors)

• used for clustering to generate clusters for input to SNP pipeline

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Salmonella Listeria Campylobacter E.coli/Shigella

Growth: Total Isolates in Pathogen Detection

2016 2017 2018

Avg. Time to Publish (hours) From Submission

Organism No. of Isolates Old SNP Pipeline New wgMLST + SNP Pipeline

Campylobacter 21,214 13 5E.coli/Shigella 52,801 23 9Listeria 19,146 14 5Salmonella 147,150 92 52

wgMLST/SNP Pipeline Comparison (hours)

• Will be covered by Arjun Prasad - Friday

• navigation panel• subtree creation• sharing of highlighted isolates• tree labeling

• Automated alerts

• first phase - stored searches send automatic notifications• second phase - 'watched' isolate mode

These are the last two major features needed to aid outbreak detection

Pathogen Browser

Requires MyNCBI login

Automated Alerts: First phase (Stored Searches)

Automated Alerts: Email Notifications

• enumerates the reason an isolate has failed for those organisms undergoing wgMLST clustering

• text file on FTP

• ftp://ftp.ncbi.nlm.nih.gov/pathogen/Results/Listeria/latest_snps/Exceptions/

• will have strain and SRA_center added as columns

exception type exception consequence lower limit upper limit actual value biosample_accAssembly validation failure Low contig N50 Not published 10000 NULL 2736 SAMN05173249Assembly validation failure High contig L50 Not published NULL 200 330 SAMN05173249Readset validation failure Insufficient coverage Not published 20 NULL 18 SAMN10079593wgMLST validation failure Too few wgMLST loci found Not clustered 2000 NULL 1723 SAMN03761820

Exceptions Report

NCBI’s Role in Combatting Antibiotic Resistance

1. Build AMR reference database (reference proteinsand hidden Markov models (HMMs) and proteinfamily hierarchy

2. Build AMRFinder tool to identify AMR proteins usingreference database

3. Use AMRFinder to identify AMR proteins in all pathogenisolates integrated into NCBI Pathogen Browser

4. Capture antibiotic susceptibility test data (AST)

5. Integrate AST into NCBI Pathogen Browser

https://ftp.ncbi.nlm.nih.gov/pub/factsheets/Factsheet_AMR_Project.pdf

From Annotation to Resistance Genes

AMRdatabase

HMMs andBLAST

Report onresistance genes

in isolate

Proteins

4,579 resistance proteins34 drug classes resisted~50% beta-lactamases565 HMMs

New beta lactamase,

qnr, and mcrsubmissions

https://www.ncbi.nlm.nih.gov/pathogens/antimicrobial-resistance/AMRFinder/

AMRFinder Alpha Release

• scientific publication describing AMRFinder and genotype/phenotype correlations on ~6K NARMS isolates

• organism-specific point mutations (29 genes) covering resistance to quinolones, macrolides, etc.

• AMR resource landing pages

• AMR Gene Browser - for every AMR gene in every isolate• starting with some high priority organisms (Klebsiella)• requires annotated genome to be available

• stress response and virulence genes

AMRFinder and Antimicrobial Resistance Resources

Pathogen Browser

Pathogen Detection Pipeline

AMRFinder and Resources

automated alerts

Sept. - Oct/2018 Nov - Dec/2018 2019

publication on AMRFinder

stress response and virulence genes

point mutations

combined protein/translated searches

AMR Gene Browser

'watched isolates'

improve time to report

begin GenBank submission

improve time to report

wgMLST for otherpathogens

wgMLST publication

pipeline publication

browser publication

NCBI Pathogen Detection Roadmap

Thank you.This research was supported by the Intramural Research Program of the NIH, National Library of Medicine.

pd-help@ncbi.nlm.nih.gov

Richa AgarwalaAzat BadretdinSlava BroverJoshua CherryJinna ChoiVyacheslav ChetverninRobert CohenMichael DiCuccioBoris FedorovMike FeldgardenLewis GeerDan HaftLianyi HanAvi KimchiMichel KimelmanWilliam KlimkeAlex KotliarovValerii LashmanovAleksandr MorgulisEyal MosesChris O'SullivanArjun Prasad

Edward RiceKirill RotmistrovskyyAlejandro A. SchafferNadya SerovaStephen SherrySergey ShiryevMartin ShumwayOleg ShutovAlexandre SouvorovTatiana TatusovaIgor TolstoyChunlin XiaoLeonid ZaslavskyAlexander ZasypkinLukas WagnerHlavina WratkoEugene Yaschenko

David LipmanJames OstellKim Pruitt

CDCFDA/CFSANGenFSUSDA-FSISPHE/FERANIHGRINIAIDWRAIRBroadWadsworth/MDHVendors: PacBio, Illumina, Roche