HIGH-RESOLUTION BACTERIAL GENOMES THROUGH PACBIO REVIO SEQUENCING OF POOLED DNA

Mauro de Medeiros Oliveira; Taís Mayumi Kuniyoshi; Iago Blanco; João Victor dos Anjos Almeida; Ricardo Pinheiro de Souza Oliveira; Alessandro Varani

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Paper Title

HIGH-RESOLUTION BACTERIAL GENOMES THROUGH PACBIO REVIO SEQUENCING OF POOLED DNA

Authors

Mauro de Medeiros Oliveira
Taís Mayumi Kuniyoshi
Iago Blanco
João Victor dos Anjos Almeida
Ricardo Pinheiro de Souza Oliveira
Alessandro Varani

Modality

Poster

Subject area

DNA and Genomics

Publishing Date

21/11/2024

Country of Publishing

Brazil | Brasil

Language of Publishing

Inglês

Paper Page

https://www.even3.com.br/anais/xmeeting-2024/824673-high-resolution-bacterial-genomes-through-pacbio-revio-sequencing-of-pooled-dna

ISBN

978-65-272-0843-3

Keywords

Pooled DNA, de novo Assembly, Lactic Acid Bacteria, PacBio REVIO Sequencing

Summary

This study investigates the high-resolution sequencing of bacterial DNA using the PacBio REVIO platform. While traditional "short-read" sequencing methods are more affordable, PacBio HiFi offers several advantages for our research: Greater Precision - HiFi sequencing generates longer reads with higher fidelity, resulting in fewer errors associated with insertions and deletions (indels) within the DNA sequence; High Coverage - Long reads enable the assembly of larger contigs (contiguous DNA segments), facilitating downstream analyses; Enhanced Efficiency - PacBio HiFi boasts faster sequencing times, requires lower DNA sample concentrations, and streamlines data processing workflows. Our FAPESP Thematic Grants research project (“Bioprospecting of bacteriocin-producing bacteria: from culture optimization to the application in animal production systems'' # 2018/25511-1) successfully isolated over 25 lactic acid bacteria from diverse production animals – pigs, poultry, and fish. Preliminary data suggests these isolates possess probiotic and/or prebiotic potential. However, sequencing the entire genome of each isolate proved cost-prohibitive, regardless of the sequencing platform. To address this challenge, we opted to construct pooled sets of genomic DNA containing DNA from various bacterial species across different genera. Five sets were created, each containing between 4 and 9 isolates. Due to its compatibility with pooled samples, the PacBio HiFi platform emerged as the most suitable choice for sequencing our samples. Sequencing yielded high-quality reads with a base quality score exceeding Q40 and an average read length surpassing 7,500 base pairs (bp). Notably, HiFi sequencing generated approximately 10 gigabase pairs (Gbp) of data per sample. The number of reads obtained varied depending on the number of isolates pooled: samples with 4 isolates yielded an average of 1.1 million reads, while samples with 9 isolates averaged 1.5 million reads. Two de novo assembly tools, Flye and HiFiAsm-meta, were employed to assemble the reads. The assemblies generated by both tools exhibited high concordance across all analyzed samples. Furthermore, for each isolate within a pool, we successfully assembled a single, circular contig encompassing the chromosomal region. Additionally, several circular contigs ranging from 12,000 bp to 120,000 bp in size were identified as plasmids originating from the various species sequenced in each pool. The assembled contigs, both chromosomal and plasmid-derived, achieved coverage values between 80x and 570x. This approach enables us to completely sequence the genomes of multiple bacterial isolates efficiently, while still retaining the advantages of high-resolution PacBio HiFi sequencing at a lower cost compared to Illumina and ONT. Sequencing this dataset with PacBio REVIO is approximately three times cheaper than using a combination of Illumina and ONT. This method not only enhances the feasibility of large-scale genomic studies but also promises to accelerate advancements in microbiological research by providing an economical solution for comprehensive genomic analysis.

Title of the Event: 20º Congresso Brasileiro de Bioinformática: X-Meeting 2024
City of the Event: Salvador
Title of the Proceedings of the event: X-Meeting presentations
Name of the Publisher: Even3
Means of Dissemination: Meio Digital

How to cite

OLIVEIRA, Mauro de Medeiros et al.. HIGH-RESOLUTION BACTERIAL GENOMES THROUGH PACBIO REVIO SEQUENCING OF POOLED DNA.. In: X-Meeting presentations. Anais...Salvador(BA) Hotel Deville Prime, 2024. Available in: https//www.even3.com.br/anais/xmeeting-2024/824673-HIGH-RESOLUTION-BACTERIAL-GENOMES-THROUGH-PACBIO-REVIO-SEQUENCING-OF-POOLED-DNA. Access in: 07/08/2025