Lab Matters Fall 2024 | Page 106

APHL 2024 POSTER ABSTRACTS

Maximizing Use of Publicly Available Genomic Data to Support Infectious Disease Outbreak Investigations

E . Smith , K . Libuit , F . Ambrosio , E . Doughty , C . Kapsak , I . Mendes , J . Otieno , M . Scribner , S . Wright , J . Sevinsky , Theiagen Genomics

The use of publicly available whole genome sequencing ( WGS ) data can provide crucial contextual information during outbreak investigations . Public health scientists can use data in NCBI to determine whether a certain publicly available genome belongs to an existing outbreak , how large that outbreak might be and investigate potential sources . However , public health laboratories often need to compare recently sequenced samples to publicly available data prior to submitting those samples to NCBI . Additionally , there are many complexities to building phylogenetic trees of bacterial pathogens , including the selection of an appropriate reference genome , calling single nucleotide polymorphisms ( SNPs ) and removing SNPs involved in homologous recombination events . Here , we describe two open-source bioinformatics workflows , Assembly _ Fetch and Snippy _ Streamline , for importing contextual genome assemblies from NCBI and performing comprehensive phylogenetic analyses . The Assembly _ Fetch workflow can import genome assemblies from NCBI to the Terra . bio platform when provided with a list of accession numbers , such as those known to be involved in a putative outbreak . The Snippy _ Streamline workflow can then be used to determine the most appropriate reference genome from a selected group of genome assemblies by calculating the centroid of the sample set using mash distances and importing that genome from the NCBI RefSeq database . Then , SNP calling is performed relative to this reference genome using Snippy and users also have the option to mask SNPs that are unlikely to have arisen from ancestral sources , such as those found within regions of homologous recombination using Gubbins . The resulting multiple-sequence alignments are used to build a high-quality maximum-likelihood phylogeny using IQTree and to calculate pairwise SNP-distances between genomes . Public health scientists can then determine whether the recently sequenced samples belong to a known outbreak , or whether they might be the result of separate transmission events . These workflows exemplify how public health laboratories can leverage publicly available WGS data to inform timely , data-driven , public health action surrounding infectious disease outbreaks .

Presenter : Emily Smith , emily . smith @ theiagen . com

Microbiome Species Profiling at Scale with the Kinnex Kit for Full-length 16S rRNA Sequencing

J . Wilkinson 1 , J . Bruand 1 , K . Chua 1 , H . Ferrao 1 , D . Lee 1 , K . Locken 2 , S . Tang 2 , E . Thai 2 , J . Sherman 2 , B . Farthing 2 , E . Tseng 1 , PacBio 1 , Zymo Research Corporation 2

Targeted 16S sequencing is a cost-effective approach for assessing the bacterial composition of metagenomic communities . This is especially true for low bacterial biomass samples where amplicon sequencing is the best option . However , the high similarity between the 16S rRNA genes of related bacteria means that sequencing the entirety of the 16S gene (~ 1.5 kb ) with high accuracy is essential for species- or strain-level characterization . Recent comparative studies have shown that PacBio full-length ( FL ) 16S sequencing outperforms other sequencing methods for taxonomic resolution and data accuracy . The Kinnex 16S rRNA kit takes amplified 16S amplicons as input and outputs a sequencing-ready library that results in an up to 12-fold throughput increase compared to standard FL 16S libraries . The Kinnex 16S kit is based on the multiplexed array sequencing ( MAS-Seq ) method ( Al ’ Khafaji et al ., 2023 ) applied to FL 16S amplicons . The result is significantly higher throughput and reduced sequencing needs for high accuracy , cost-effective FL 16S sequencing with the ability to multiplex up to 1,536 amplicon samples per SMRT Cell . We tested the Kinnex 16S rRNA kit on a diverse range of samples ( 13 types ) including mock communities , feces , skin swab , plant , veterinary wound swab , soil , vaginal swab , rhizosphere and wastewater sludge . We then analyzed the data using a user-friendly bioinformatics pipeline , HiFi-16S-workflow , that provides a FASTQ-to-report analysis solution for FL 16S HiFi reads . The results show that Kinnex 16S sequencing can yield > 30k average reads per sample at a 1,536-plex on a single Revio SMRT Cell or at a 768-plex on a Sequel IIe SMRT Cell . Comparing Kinnex 16S to standard FL 16S datasets , we found a high correlation and no bias in community compositions and were able to assign up to ~ 99 % of denoised reads to species . In addition , because of the higher number of reads per sample , Kinnex 16S allows for more recovery of lower abundance species . With the Kinnex 16S rRNA kit , researchers may now multiplex more samples to dramatically reduce cost per sample or to profile each sample deeper with more reads / sample . The additional reads / sample along with better taxonomic resolution is advantageous for numerous environmental sample types which are often highly diverse , containing many microbial species .

Presenter : Jeremy Wilkinson , jwilkinson @ pacb . com

Mini-meta Study : Surface , Air , Water and Food Metagenomics Workflow Using the Automated Concentrating Pipette Select

D . Alburty , InnovaPrep

Metagenomics is an expanding field of public health and the number of published studies is rapidly increasing . Environmental compartments relevant to human health include surfaces , air , water / wastewater and food . While these compartments are loosely connected , the sample collection and processing methods are different for each . Sample matrix preparation , including target preconcentration , must usually be performed prior to metagenomic methods such as shotgun metagenomics , whole genome sequencing , microarrays and other advanced molecular techniques . Four studies ( public and academic ), each representative of an environmental compartment , are compared and contrasted with regard to the sample collection , automated sample preparation and analytical workflow .

Presenter : David Alburty , dalburty @ innovaprep . com