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Scientific posters

ASM 2024  |  2024

An Integrated Approach for Pathogen Detection, AMR Monitoring, and Functional Analysis in Wastewater

X. Cheng1, J. Wilkinson2, K. Ngo2, P. Baybayan2, Y. Kim2, P. Pham2, E. Carrasco1, S. Tang1, J. Shen1, and K. Locken1, Zymo Research Corporation1, Pacific Biosciences of California, Inc.2

Wastewater surveillance has emerged as a valuable epidemiological instrument in public health. In this study, we introduce an innovative, integrated methodology for the concurrent detection of pathogens, monitoring of antimicrobial resistance (AMR), and functional analysis in wastewater. Leveraging the advanced sample preparation solutions provided by Zymo Research and PacBio Onso short-read sequencing, we aimed to enhance our understanding of microbial dynamics in wastewater and provide a report for actionable insights for public health and water treatment facilities. Real wastewater samples from local treatment facilities were processed using Zymo Research nucleic acid purification technologies. The PacBio Onso short-read sequencing system, which yields Q40+ accuracy, captured a comprehensive microbial profile. Metagenomic sequence data was downsampled to 8 million reads per sample to be comparable to data obtained using the Illumina NextSeq 2000 system at the same depth, then analyzed using the Zymo wastewater analysis pipeline. By comparing the number of taxa, functional groups, and AMR, this study shows the potential of the high accuracy short-read PacBio Onso system for wastewater surveillance. The outcomes of this study offer multifaceted benefits for public health departments and water treatment facilities. Accurate pathogen detection enables the prediction of potential disease outbreaks, empowering public health authorities to implement proactive measures. AMR monitoring provides crucial insights into resistance gene prevalence, informing strategies against the spread of antibiotic resistance. Functional analysis delves deeper into the intricacies of microbial communities within wastewater, specifically highlighting Nitrogen removal species, Phosphorus-accumulating organisms (PAOs), Methylotrophs, Filamentous bacteria, and pathogens. This nuanced understanding empowers water treatment facilities to customize effective strategies, optimizing processes for contaminant removal and water safety. This research signifies a significant stride in proactive public health management and water treatment process optimization for enhanced environmental and public well-being.
ASM 2024  |  2024

Long-read metagenome assembly produces hundreds of high-quality MAGs from wetland soil

Daniel M. Portik1, Luis E. Valentin-Alvarado2,3, Jeremy E. Wilkinson1, Jillian F. Banfield2 1. PacBio, 1305 O’Brien Dr, Menlo Park, California 93025 USA 2. Innovative Genomics Institute, University of California, Berkeley, California 94720 USA 3. Department of Plant and Microbial Ecology, University of California, Berkeley, California USA

Long-read sequencing has revolutionized metagenome assembly, overcoming many historical challenges. New metagenome assembly algorithms have been designed specifically for PacBio HiFi reads, which take advantage of high read accuracy (>Q20). These methods, including hifiasm-meta and metaMDBG, routinely produce long, circular contigs that represent complete bacterial and archaeal genomes. Following assembly, long-read-specific binning workflows can be used to identify metagenome-assembled genomes (MAGs) from the assembled contigs. The combination of new HiFi assembly methods and long-read binning workflows dramatically increases the number and quality of MAGs obtained. We demonstrate the power of these approaches by performing HiFi sequencing for a wetland soil sample on the Revio system. Our analysis resulted in over 1,200 MAGs, including more than 500 high-quality MAGs. A majority of MAGs were assigned to Bacteria, predominantly Acidobacteria, but also included representatives from many understudied taxa. Our genome-centric analysis unveiled a diverse and abundant archaeal presence in the deeper layers of seasonally inundated wetland soil. We successfully reconstructed complete genomes for representative lineages of many archaeal groups, including Asgard Archaea, Bathyarchaeia, Thaumarchaeota, Hadearchaeales, Methanomethyliales, and Micrarchaeia, along with their integrated and coexisting extrachromosomal elements (ECEs). Our findings underscore the importance of complete genomes in metabolic analysis and the identification and characterization of novel ECEs that contribute to genomic diversity and facilitate horizontal gene transfer. These results enhance our understanding of soil archaeal evolution. Furthermore, they provide valuable insights for future studies aiming to exploit the potential of elusive mobile genetic elements for editing uncultivated species in microbial communities.
ASM 2024  |  2024

Microbiome species profiling at scale with the Kinnex kit for full-length 16S rRNA sequencing

Jeremy E Wilkinson1, Jocelyne Bruand1, Khi Pin Chua1, Heather Ferrao1, Davy Lee1, Kristopher Locken2, Shuiquan Tang2, Ethan Thai2, John Sherman2, Brett Farthing2, Elizabeth Tseng1 1. PacBio, 1305 O’Brien Drive, Menlo Park, CA, USA 94025 2. Zymo Research Corporation, 17062 Murphy Avenue, Irvine, CA, USA 92614

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.
ASM 2024  |  2024

New long-read metagenome assembly methods increase the number of high-quality MAGs from host and environmental microbiomes

Daniel J. Nasko1, Jeremy E. Wilkinson1, Daniel M. Portik1 1. PacBio, 1305 O’Brien Dr, Menlo Park, California 93025 USA

There are many challenges involved with metagenome assembly, including the presence of multiple species, uneven species abundances, and conserved genomic regions that are shared across species. Many of these historical challenges can be overcome using long-read sequencing. In particular, highly accurate PacBio HiFi reads generated from the Sequel IIe and Revio systems can provide major advantages for metagenome assembly. New metagenome assembly algorithms have been designed specifically for HiFi reads, including hifiasm-meta and metaMDBG, which take advantage of this high read accuracy. These algorithms are capable of producing long, circular contigs which represent complete bacterial or archaeal genomes. Long-read specific binning workflows, such as the HiFi-MAG-Pipeline, can be used to process the assemblies in order to extract metagenome assembled genomes (MAGs). The combination of new HiFi assembly methods and long-read binning workflows improves the total number of MAGs obtained, while also improving the completeness and contiguity of individual MAGs. We demonstrate the power of using these approaches for a variety of microbiomes. We show that more complete, circular MAGs are routinely produced from HiFi metagenome assemblies. We find metaMDBG results in up to a 200% increase in total MAGs, relative to hifiasm-meta. It also produces more small (i.e. <200 Kbp) circular contigs that likely represent plasmids, viruses, and other mobile elements. Overall, we demonstrate that HiFi sequencing can be used to obtain many high-quality MAGs from a variety of microbiomes.
ESHG 2024  |  2024

Assessment of read depth requirements for gene and isoform discovery: a comparative study of long-read and short-read RNA sequencing data in human heart and brain

Nina Gonzaludo*1, Jocelyne Bruand1, Amy Klegarth1, Jason Underwood1, Elizabeth Tseng1, Birth Defects Research Laboratory2, Kimberly A. Aldinger3 1. PacBio, Menlo Park, CA, USA, 2. University of Washington, Seattle, WA, USA, 3. Seattle Children’s Research Institute, Seattle, WA, USA

The PacBio full-length Kinnex RNA kit provides complete transcript coverage for isoform and gene discovery in tissues of interest, enabling understanding of biology and disease. With Kinnex, fewer long reads are needed for gene discovery compared to shortread RNA seq. The majority of known genes and isoforms can be discovered using full-length Kinnex kits at 10-20M reads per sample, suggesting multiplexing may be a cost-effective yet comprehensive option.
ESHG 2024  |  2024

PureTarget: An amplification-free workflow for genetic and epigenetic profiling of short tandem repeat expansions

Guilherme De Sena Brandine, Valeriya Gaysinskaya, Janet Aiyedun, Julian Rocha, Duncan Kilburn, Sarah Kingan, Egor Dolzhenko, Zoi Kontogeorgiou, Anita Szabo, Christina Zarouchlioti, Robert Thaenert, Fabio Fuligni, Aidan Hennigan, Chelsea Roselund, Alesia Piselli, Pilar Alvarez Jerez, Kimberley Billingsley, Sonia Lameiras, Sylvain Baulande, Petra Liskova, Alice Davidson, Georgios Koutsis, Georgia Karadima, Stéphanie Tomé, Michael Eberle

Background/Objectives: Short tandem repeats (STRs) are DNA sequences composed of multiple copies of 1-6bp motifs. STRs are ubiquitous in the human genome, and some are prone to expansions that cause disease. Many pathogenic STR expansions are variable in length and sequence composition, both at the individual and population scale. Motif composition, sequence length and CpG methylation often dictate disease onset and severity. An ideal approach to identifying pathogenic STR expansions requires accurate assessments of all these properties in a single assay. Methods: We describe a robust amplification-free protocol to generate long-read HiFi sequencing libraries containing a panel of loci associated with 20 pathogenic STR expansions. The protocol can be multiplexed to sequence up to 48 samples at 20 to 1000x coverage per locus in one PacBio sequencing run. We measured accuracy in 129 samples with validated pathogenic expansions at loci including CNBP, DMPK, RFC1 and C9orf72. We tested 1720 sample-expansion combinations, including technical replicates, for expansions between 66bp and >10kb. Results: Our assay correctly categorized all (129/129) expansions, including the detection of hypermethylation in the FMR1 expansion and differentiating the pathogenic AAGGG motif in RFC1. We identified additional expansions in FXN, RFC1 and TCF4, consistent with these loci having carrier frequencies between 1:50 and 1:20. Excluding these three genes, we found no unexpected expansions (0/2193) in any sample/loci combinations. Conclusions: The protocol provides an accurate description of the tissue-level molecular landscape of various pathogenic STRs and is adaptable to other loci in the human genome.
AACR 2024  |  2024

Improved detection of low frequency mutations in ovarian and endometrial cancers by utilizing a highly accurate sequencing platform

T. Revil1, N. Pezeshkian2, L. Gilbert1, A. Sockell2, J. Ragoussis1; 1) McGill University, Quebec, QC, Canada, 2) PacBio, Menlo Park, CA

Ovarian and endometrial cancers come within the top-4 for incident cancers as well as deaths in North American women. Cure rates have not improved in 30 years as high-grade subtypes continue to be diagnosed in Stage III/IV. Attempts at early diagnosis have failed because high-grade cancer cells exfoliate and metastasize while the primary cancer is small and undetectable by existing tests based on imaging and blood-based tumor markers. DOvEEgene (Detecting Ovarian and Endometrial cancers Early using genomics) is a genomic uterine pap test developed by a McGill team to screen and detect these cancers while they are confined to the gynecologic organs and curable by surgery. The test identifies pathogenic somatic mutations in uterine brush samples. Here we tested the Onso system, a highly accurate sequencing technology from PacBio in order to potentially increase sensitivity while driving down sequencing costs by reducing required sequencing depth vs the current NGS standard. A highly sensitive, error-correcting capture technology (DOvEEgene-SureSelectHS) utilizing duplex error correction sequencing interrogated the exons of 23 genes involved in the development of sporadic and hereditary ovarian and endometrial cancers. We applied a combination of germline gene panel testing on saliva samples with deep duplex sequencing to detect somatic mutations at <0.1% VAF, interrogation of microsatellite loci for instability, and low coverage WGS for copy number analysis of uterine brush samples. We sequenced 20 duplex Illumina sequencing libraries produced using the DovEE assay at PE 100bp mode and compared Onso data in non- duplex sequencing mode as well as duplex sequencing mode to the original Illumina duplex sequencing method. Here, we present this comparison and highlight the benefits of high accuracy sequencing for the detection of very low frequency (<0.1%) somatic mutations. We observed improved mismatch rates for Onso data compared to Illumina, even after duplex error correction was applied. In addition, we found that more individuals are called as displaying microsatellite instablity from the Onso data, which may be due to improved sequencing performance in repetitive regions for Onso. Finally, we observed fewer potential false positive variant calls in the Onso data, highlighting the value of improved sequencing accuracy for rare variant detection.
AACR 2024  |  2024

Improved liquid biopsy assay performance using sequencing by binding (SBB) on the Onso system

D. Nasko, P. Pham, S. Joshi, K. Kim, N. Pezeshkian, Y. Kim, A. Sockell, J. Korlach; PacBio, Menlo Park, CA

Liquid biopsy is revolutionizing the field of early cancer detection research through non-invasive detection of tumor DNA in the blood. However, existing liquid biopsy assays are limited in their sensitivity for ctDNA detection at low variant allele frequencies (VAFs), with most relying on extreme sequencing depth and computational error correction to separate the true ctDNA signal from background errors. This limitation is particularly problematic in the area of early cancer detection, in which expected ctDNA allele frequencies are extremely low. Novel strategies are therefore needed to help improve liquid biopsy assay sensitivity and reduce per-sample sequencing requirements. Here we describe PacBio’s application of the Onso short-read sequencing system to enable detection of ctDNA at low VAFs using the SeraCare Complete ctDNA Mutation Mix reference standard. The Onso system makes use of a novel sequencing by binding (SBB) method to achieve up to 15x greater quality scores, with ≥90% of reads at Q40 or above. We performed targeted capture and sequencing of libraries prepared from the SeraCare reference mix diluted into WT human DNA at the following VAFs: 0.00% (WT), 0.05%, 0.10%, 0.25%, and 0.50%, and compared the sensitivity at each VAF for SBB compared to a competitor method using sequencing by synthesis (SBS) at varying sequencing depths. We observed superior sensitivity for ctDNA detection at low VAFs (0.05%, 0.1%) using SBB at half the sequencing depth compared to SBS, in part due to reduced false positive calling in the WT sample for SBB. Furthermore, SBB was able to achieve comparable sensitivity results to SBS using four-fold less sequencing, and without the use of computational error correction. Finally, combining SBB with computational error-correction methods boosted sensitivity even further, suggesting an additive value for these technologies. Taken together, our results demonstrate the potential of SBB to improve upon existing methods of liquid biopsy and better enable research on early cancer detection.
AGBT 2024  |  2024

Building the spectrum of ground truth genetic variation in a four-generation 28-member CEPH family

Zev N Kronenberg1, Katherine M Munson2, David Porubsky2, Cillian Nolan1, William J Rowell1, Brent S Pedersen3, Cairbre Fanslow1, Primo Baybayan1, Nidhi Koundinya2, William Harvey2, Kendra Hoekzema2, Jordan Knuth2, Gage Garcia2, Tom Mokveld1, Egor Dolzhenko1, Scott Watkins3, Deborah W Neklason3, Aaron R Quinlan3, Lynn B Jorde3, Evan E Eichler2 and Michael A Eberle1 1) PacBio, 1305 O’Brien Drive, Menlo Park, CA 94025 2) University of Washington, Seattle WA, 98195 3) University of Utah, Salt Lake City, UT, 84114

Comprehensive ground truth data is required for validating sequencing pipelines in clinical settings, assessing the strengths and weaknesses of genome sequencing technologies, and improving variant detection software. Truth sets of genomic variation have lagged advances in sequencing accuracy and completeness; furthermore, current truth sets are confined to easy-to-characterize regions that comprise ~80% of the human genome. These benchmarking sets are mostly limited to small variants, missing complex variants like SVs and mega-bases of tandem repeat variation. Developing more complete truth sets will spur improvement in variant calling algorithms in under-characterized regions of the genome. We have built a new truth set, leveraging the power of a large four generation 28-member family (CEPH 1463) using multiple sequencing technologies (PacBio HiFi and SBB, ONT, Illumina, and Strand-seq). Nearly all of these samples are derived from blood thus eliminating the problem of cell line artifacts. Using ensemble-based variant calls from read mapping and long-read genome assembly, we built a highly sensitive variant call set spanning the spectrum of variant size and complexity. From these calls, we built haplotypes and mapped recombination events among 10 second and third generation family members (two parents and eight children). Integrating the high-resolution haplotype map with multiple variant callers across sequencing technologies, we have built a truth set for a ten-member pedigree. In total, our pedigree-validated truth set contains 5,023,261 SNVs, 1,009,108 indels and 20,572 structural variants totaling 16 Mb of genetic variation. By using inheritance patterns to validate the accuracy of the variant calls, this benchmarking database combines the strengths of the different technologies increasing the number of small variants by 14% and 6% in NA12878 compared to the Genome in a Bottle and Platinum Genomes, respectively. Compared to GIAB’s NA12878, we have expanded the high quality regions from 82.9% of the genome to 91.4%. Using technical replicates, we evaluate the accuracy of different sequencing technologies and variant callers against this comprehensive dataset. The full sequencing data and validated variants identified in this study will be publicly available to serve as a valuable community resource, as the largest multi-generational pedigree sequenced with long-read technologies.
AGBT 2024  |  2024

Resolving variation in polymorphic regions of the human genome

Egor Dolzhenko1, Graham S Erwin2, Katherine Wang2, Zev Kronenberg1, William J Rowell1, Anna C Ferrari3, Garrison Pease3, Daniel Schwartz3, Benjamin Gartrell3, Ahmed Aboumohamed3, Alex Sankin3, Pedro Maria3, Kara Watts3, John M Greally4, Patrick Wilkinson5, Yashoda Rajpurohit5, John Loffredo5, Denis Smirnov5, Manuel A Sepulveda5, Charles G Drake5, Alex Robertson1, Michael P Snyder2, Michael A Eberle11. PacBio, Menlo CA, USA; 2. Stanford University,CA, USA; 3. Montefiore-Einstein Cancer Center, NY, USA; 4. Einstein Epigenomics Center, NY, USA; 5. Janssen Research and Development LLC, PA, USA

The human genome contains thousands of repeat-rich polymorphic regions whose structure has not been systematically described. These regions produce large collections of variant calls sometimes called variation clusters. Variation clusters are typically excluded from tertiary analysis because it is difficult to interpret and catalog them. One example is the 3.5 Kbp region in an intron of the KCNMB2 gene which contains over 30 constituent simple repeats that jointly create many insertions, deletions, and mismatches in alignments of reads over this region. The corresponding variant calls are often incorrectly prioritized as potentially pathogenic, requiring significant resources to curate and rule out. To address these issues, we propose a novel computational framework to systematically detect, annotate, and catalog variation clusters. A distinguishing characteristic of our approach relative to traditional variant calling methods, is the ability to annotate and resolve entire regions of high sequence polymorphism as single units instead of fragmenting them into variation clusters. These regions can be subsequently genotyped using the recently developed tandem repeat genotyping tool (TRGT). We show that our method can accurately detect reference coordinates and resolve structures of KCNMB2, MUC1, CEL, INS and 20 other medically relevant variable number tandem repeats. Using real and simulated data we also show that our method can locate and call pathogenic expansions of 50 disease-causing repeats and nine polyalanine repeats composed of highly variable motif sequences. To demonstrate the usefulness of our method for cancer genome studies, we applied it to normal, polyp, and adenocarcinoma PacBio HiFi samples originating from the same individual and identified a tandem repeat that progressively expands in length from normal to polyp to adenocarcinoma samples in the 5′ UTR of LIMD1, a reported tumor suppressor gene. To further highlight our ability to resolve variation we characterized differences in repeat composition and methylation between three prostate tumors and their normal counterparts and also a panel of 100 unrelated genomes. To make all these analyses accessible to other genome researchers, we are releasing a learning resource with tutorials describing how to catalog variation in the polymorphic regions of the human genome in publicly available PacBio HiFi samples.
PAG 2024  |  2024

A high-throughput, low-cost automated library prep method for PacBio long-read sequencing at scale

Gregory Young1, Aurelie Souppe1, Kaitlyn Scott1, Davy Lee1, Gloria Diaz1, Lin Wang1, Nethmi Gunathilake1, and Greg Concepcion1 1. PacBio, 1305 O’Brien Drive, Menlo Park, CA 94025

Bottlenecks in long-read library prep workflows such as DNA shearing (aka fragmentation) and size selection are barriers to scaling long-read sequencing (LRS) independent of sequencing costs. Here we present a new automated highthroughput library prep method for PacBio native long-read sequencing that removes these bottlenecks, dramatically lowers costs, and operates in a 96-well plate format.
PAG 2024  |  2024

High-throughput HMW DNA animal blood extraction and sequencing on the PacBio Revio system

Deborah Moine1, Adam Bates3, Michelle Kim2, Jacob Brandenburg1, Nadia Sellami1, Jackson Mingle2, Jeffrey Burke2, Julian Rocha2, Aurelie Souppe1,Heather Ferrao1,Gregory Concepcion1, Caroline Howard3,and Kelvin J Liu2 1. PacBio, 1305 O’Brien Drive, Menlo Park, CA 94025, 2. PacBio, 701 E. Pratt Street, Baltimore, MD 21202, 3. Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK

The Darwin Tree of Life project is a large biodiversity initiative aiming to generate high quality genomes for 70,000 species of eukaryotes across Britain and Ireland. For large-scale projects such as these, high throughput (HT) solutions are critical; PacBio Nanobind HT DNA extraction kits combined with the Revio system address these needs by significantly increasing throughput and lowering the cost of long-read sequencing. We present fully automated methods for HT DNA extraction, shearing, library preparation, and PacBio HiFi sequencing of blood from animals with nucleated and non-nucleated red blood cells (nRBCs). These workflows can prepare 96 samples from DNA extraction to libraries that are ready for loading in ~10 hours. High molecular weight (HMW) DNA is extracted using the Nanobind disks on the Thermo Fisher KingFisher APEX or Hamilton NIMBUS Presto automated systems. Using these workflows, we can recover ~5−20 μg of dsDNA per extraction on a 96-well plate in 2.5 hours. The HMW DNA is size selected in a 96-well plate using the PacBio SRE kit and sheared to 15−20 kb by pipetting on an automated liquid handler. Last, libraries are prepared on the fully automated Hamilton NGS Star. The methods presented here utilize standard configurations of Hamilton instruments and can easily be incorporated into existing workflows. A single Revio SMRT Cell typically generates sufficient HiFi coverage for high-quality de novo assembly of a diploid vertebrate genome.
PAG 2024  |  2024

Increasing the throughput of full-length 16S sequencing with Kinnex kits

Jeremy E Wilkinson, Khi Pin Chua, Siyuan Zhang, Jason Underwood, Minning Chin, Wei-Shen Cheng, Sian Loong Au, Primo Baybayan, Holly Ganz, Guillaume Jospin, Ye Tao, Qin Lin, Elizabeth Tseng

In the past several years, the ability to capture the full-length (FL) 16S rRNA gene with PacBio HiFi sequencing has enabled researchers to profile microbiomes in significantly higher resolution. Only full-length and highly accurate 16S sequences can robustly identify the broad range of bacteria seen in complex microbial communities at the species level, without bias. To further increase the cost effectiveness of FL 16S sequencing, we applied the Kinnex 16S rRNA kit, which is based on the multiplexed array sequencing (MAS-Seq) method (Al’Khafaji et al., 2023), to FL 16S amplicons. The MAS-Seq method is a versatile throughput increase method that takes advantage of the longer HiFi read lengths to concatenate amplicons into ordered arrays with programmable array sizes. We demonstrated that Kinnex 16S results in an ~8–12-fold throughput increase compared to standard FL 16S. We tested the method on a diverse range (11 types) of samples including mock communities, human and animal feces/guts, soil, sediment, rhizosphere, sludge, and water. 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. Comparing the Kinnex 16S to standard FL 16S datasets, we found no bias in community compositions and were able to assign up to ~90–99% of denoised reads to species. In addition, on the highly complex ZymoBIOMICS Fecal Reference with TruMatrix Technology (D6323) sample, we found Kinnex 16S to have high correlation to taxonomic abundances estimated from shotgun metagenomics sequencing using the same sample, emphasizing that it’s possible to get shotgun metagenome taxonomic resolution at amplicon sequencing costs with FL 16S HiFi sequencing. Furthermore, with Kinnex 16S, researchers may now multiplex more samples to reduce cost per sample or to profile each sample deeper with more reads per sample.
PAG 2024  |  2024

New long-read metagenome assembly methods increase the number of high-quality MAGs from host-associated microbiomes

Daniel M. Portik, Jeremy E. Wilkinson

There are many challenges associated with metagenome assembly, which include: the presence of multiple species uneven and unknown species abundances conserved genomic regions shared across species strain-level variation within species PacBio HiFi sequencing produces highly accurate long reads (>Q20, >99% accuracy) which provide major advantages for metagenome assembly. New metagenome assembly algorithms have been developed specifically for HiFi reads, including hifiasm-meta1 and metaMDBG.2 These methods make it possible to reconstruct full metagenome-assembled genomes (MAGs) for many high abundance species. However, discontiguous assemblies will occur for lower abundance taxa. Post-assembly tools incorporating binning methods are required to identify and extract additional MAGs. The HiFi-MAG-Pipeline (v2) is a comprehensive workflow for processing long-read assemblies, and includes major steps such as binning, quality filtering, and taxonomic identification. Here, we demonstrate the performance of these methods using a variety of HiFi metagenomic datasets.
PAG 2024  |  2024

Scalable, cost-effective isoform sequencing with Kinnex full-length RNA kit using long-read sequencing

Jacob Brandenburg1, Elizabeth Tseng1, Heather Ferrao1, Jocelyne Bruand1, Armin Toepfer1, Gloria Sheynkman2, Madison Mehlferber2, Vasilli Pavelko2, 1. PacBio, 1305 O’Brien Drive, Menlo Park, CA 94025 2. Dept of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville VA 22093

The Kinnex full-length RNA kit takes total RNA as input and outputs a sequencing-ready library that results in an 8-fold throughput increase compared to typical Iso-Seq libraries. Combined with the Iso-Seq analysis in SMRT Link software, PacBio offers costeffective isoform sequencing that does not require orthogonal sequencing methods.
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