From Forensics to Clinical Research: Expanding the Variant Calling Pipeline for the Precision ID mtDNA Whole Genome Panel

Despite a multitude of methods for the sample preparation, sequencing, and data analysis of mitochondrial DNA (mtDNA), the demand for innovation remains, particularly in comparison with nuclear DNA (nDNA) research. The Applied Biosystems™ Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific, USA) is an innovative library preparation kit suitable for degraded samples and low DNA input. However, its bioinformatic processing occurs in the enterprise Ion Torrent Suite™ Software (TSS), yielding BAM files aligned to an unorthodox version of the revised Cambridge Reference Sequence (rCRS), with a heteroplasmy threshold level of 10%. Here, we present an alternative customizable pipeline, the PrecisionCallerPipeline (PCP), for processing samples with the correct rCRS output after Ion Torrent sequencing with the Precision ID library kit. Using 18 samples (3 original samples and 15 mixtures) derived from the 1000 Genomes Project, we achieved overall improved performance metrics in comparison with the proprietary TSS, with optimal performance at a 2.5% heteroplasmy threshold. We further validated our findings with 50 samples from an ongoing independent cohort of stroke patients, with PCP finding 98.31% of TSS’s variants (TSS found 57.92% of PCP’s variants), with a significant correlation between the variant levels of variants found with both pipelines.

511. Treatment with Molnupiravir in the MOVe-In and MOVe-Out Clinical Trials Results in an Increase in Transition Mutations Across the SARS-CoV-2 Genome

Background Molnupiravir (MOV), (MK-4482, EIDD-2801) is being clinically developed for the treatment of COVID-19 disease caused by SARS-CoV-2. MOV is the orally administered 5′-isobutyrate prodrug of the active, antiviral ribonucleoside analogue, N-hydroxycytidine (NHC, EIDD-1931) which inhibits viral replication by induction of mutations in the viral genome, leading to viral error catastrophe. In 2 clinical studies, hospitalized (MOVe-In) and non-hospitalized (MOVe-Out) participants were treated for 5 days with MOV and followed up to Day 29. Viral RNA isolated from nasal swab samples were sequenced to determine the rate, distribution and type of viral mutations observed after MOV treatment. Methods RNA isolated from nasopharangeal swab samples collected during study conduct was quantified by RT-PCR. Samples containing >22,000 copies/mL of RNA underwent complete genome NGS using the Ion AmpliSeq SARS-CoV-2 research panel and Ion Torrent sequencing. Mutation rates were calculated by determining the number of nucleotide changes observed across the entire genome at Day 3 and/or Day 5 compared to baseline. Results Combined data from both studies showed an increase of ~2-4 fold in the viral mutation rate post-baseline in MOV treated compared with placebo. Mutations were distributed across the entire genome with only a minority being observed in more than one sample. The most frequent mutations were transitions of C to U observed in the highest MOV dose group (800 mg/BID). Conclusion Consistent with the proposed mechanism of action of MOV, an increase in the rate of transition mutations in the virus was observed in post-baseline nasal swab samples from participants treated with MOV compared with placebo. Disclosures Julie Strizki, PhD, Merck & Co., Inc. (Employee, Shareholder) Jay Grobler, PhD, Merck & Co., Inc. (Employee, Shareholder) Ying Zhang, PhD, Merck & Co., Inc. (Employee, Shareholder) Jiejun Du, PhD, Merck & Co., Inc. (Employee, Shareholder) Shunbing Zhao, PhD, Merck & Co., Inc. (Employee, Shareholder) Diane Levitan, PhD, Merck & Co., Inc. (Employee, Shareholder) Alex Therien, PhD, Merck & Co., Inc. (Employee, Shareholder) Joan R. Butterton, MD, Merck Sharp & Dohme Corp. (Employee, Shareholder) Nicholas Murgolo, PhD, Merck & Co., Inc. (Employee, Shareholder)

Seven Complete Chloroplast Genomes from Symplocos: Genome Organization and Comparative Analysis

In the present study, chloroplast genome sequences of four species of Symplocos (S. chinensis for. pilosa, S. prunifolia, S. coreana, and S. tanakana) from South Korea were obtained by Ion Torrent sequencing and compared with the sequences of three previously reported Symplocos chloroplast genomes from different species. The length of the Symplocos chloroplast genome ranged from 156,961 to 157,365 bp. Overall, 132 genes including 87 functional genes, 37 tRNA genes, and eight rRNA genes were identified in all Symplocos chloroplast genomes. The gene order and contents were highly similar across the seven species. The coding regions were more conserved than the non-coding regions, and the large single-copy and small single-copy regions were less conserved than the inverted repeat regions. We identified five new hotspot regions (rbcL, ycf4, psaJ, rpl22, and ycf1) that can be used as barcodes or species-specific Symplocos molecular markers. These four novel chloroplast genomes provide basic information on the plastid genome of Symplocos and enable better taxonomic characterization of this genus.

Genotype May Influence Bacterial Diversity in Bark and Bud of Vitis vinifera Cultivars Grown under the Same Environment

Viticulture is globally an important economic activity, and grapevine microbiomes hold a significant role in influencing yield and quality. Earlier studies showed that cultivar and agronomic management affect grapevine microbiome structure and, potentially, the quality of the end product. While microbial dynamics and ecology were established on some grapevine tissues, i.e., leaves and grapes, there is less knowledge deciphering microbiomes on other tissues, i.e., barks and buds. Moreover, although the impact on the microbiome of the so-called “vitivinicultural terroir” is well established, there are limited data considering microbiomes of genetically diverse cultivars within the same environment. Our study aims to explore microbiome diversity on bud and bark tissues of 37 different grapevine cultivars under the same environment and agronomic management. We targeted the V2-9 regions of the 16S rRNA gene of the microbiomes in bark and buds at the onset of new vegetation and bud expansion using Ion Torrent sequencing technology. Our results show that these tissues display high bacterial diversity regardless of cultivars’ use. Proteobacteria, Bacteroidetes, and Actinobacteria were the most prevalent among 11 detected phyla. The genotype of the cultivar seems to affect bacterial diversity and structure (p < 0.001) within the same environment. Our approach highlights the efficiency of high-throughput sequencing to unfold microbiomes of several grapevine parts that could be an important source of microbial inoculation and an important molecular fingerprint of the wine and grape end products.

IonCRAM: a reference-based compression tool for ion torrent sequence files

Background Ion Torrent is one of the major next generation sequencing (NGS) technologies and it is frequently used in medical research and diagnosis. The built-in software for the Ion Torrent sequencing machines delivers the sequencing results in the BAM format. In addition to the usual SAM/BAM fields, the Ion Torrent BAM file includes technology-specific flow signal data. The flow signals occupy a big portion of the BAM file (about 75% for the human genome). Compressing SAM/BAM into CRAM format significantly reduces the space needed to store the NGS results. However, the tools for generating the CRAM formats are not designed to handle the flow signals. This missing feature has motivated us to develop a new program to improve the compression of the Ion Torrent files for long term archiving. Results In this paper, we present IonCRAM, the first reference-based compression tool to compress Ion Torrent BAM files for long term archiving. For the BAM files, IonCRAM could achieve a space saving of about 43%. This space saving is superior to what achieved with the CRAM format by about 8–9%. Conclusions Reducing the space consumption of NGS data reduces the cost of storage and data transfer. Therefore, developing efficient compression software for clinical NGS data goes beyond the computational interest; as it ultimately contributes to the overall cost reduction of the clinical test. The space saving achieved by our tool is a practical step in this direction. The tool is open source and available at Code Ocean, github, and http://ioncram.saudigenomeproject.com.

High-Quality Resolution of the Outbreak-Related Zika Virus Genome and Discovery of New Viruses Using Ion Torrent-Based Metatranscriptomics

Arboviruses, including the Zika virus, have recently emerged as one of the most important threats to human health. The use of metagenomics-based approaches has already proven valuable to aid surveillance of arboviral infections, and the ability to reconstruct complete viral genomes from metatranscriptomics data is key to the development of new control strategies for these diseases. Herein, we used RNA-based metatranscriptomics associated with Ion Torrent deep sequencing to allow for the high-quality reconstitution of an outbreak-related Zika virus (ZIKV) genome (10,739 nt), with extended 5′-UTR and 3′-UTR regions, using a newly-implemented bioinformatics approach. Besides allowing for the assembly of one of the largest complete ZIKV genomes to date, our strategy also yielded high-quality complete genomes of two arthropod-infecting viruses co-infecting C6/36 cell lines, namely: Alphamesonivirus 1 strain Salvador (20,194 nt) and Aedes albopictus totivirus-like (4618 nt); the latter likely represents a new viral species. Altogether, our results demonstrate that our bioinformatics approach associated with Ion Torrent sequencing allows for the high-quality reconstruction of known and unknown viral genomes, overcoming the main limitation of RNA deep sequencing for virus identification.

Transcriptome analysis of oil palm (Elaeis guineensis Jacq.) roots under waterlogging stress

Waterlogging seriously constrains growth and yields in oil palm. To date, the responsive molecular changes caused by waterlogging in oil palm remain elusive. To elucidate the molecular genetic mechanisms of waterlogging stress, two varieties of oil palm Deli x Lamé and Deli x Ghana were used. The transcriptome profiles of the roots under waterlogging stress and normal conditions were compared via Ion Torrent Sequencing. Four libraries (GNR, GSR, SNR, and SSR) of oil palm roots after 45 days of normal watering and waterlogging stress were constructed. Approximately 6.2 million sequenced reads per library were obtained, with 5.5 million mapped reads (88.64%) similar to the oil palm genome in the GenBank database. A comparison of GNR/GSR showed a high of 3,289 DEGs with most genes up-regulated (1,863 DEGs). The GO analysis revealed the distribution of the DEGs among various pathways, suggesting a wide spectrum of physiological processes impacted by waterlogging stress. Moreover, qRT-PCR showed strong expression of all selected RNA-seq genes in waterlogged Deli x Ghana (GSR), especially GST, SAPK10 and NAC29 that are reported for the time to respond to waterlogging stress. Thus, this study not only reveals the comprehensive mechanisms of waterlogging responsive transcription in oil palm, but also establishes Deli x Ghana as a highly-adaptable variety to waterlogging conditions.