Profiling of Human Gut Virome with Oxford Nanopore Technology

Abstract

<jats:title>Abstract</jats:title><jats:p>Human gut virome play critical roles in maintaining gut microbial composition and functionality, as well as host physiology and immunology. Yet, there are insufficient amount of studies on this topic mainly due to methodological limitations, including enrichment of viruses (phages and host viruses) as well as short read-length from current sequencing technology. Here we developed a full working protocol for analyzing human gut virome using physical enrichment, reverse transcription and random amplification, and eventually the state-of-art single-molecule real-time sequencing (SMRT) platform of Oxford Nanopore Technology (ONT). We demonstrate that sequencing viral DNA directly, or viral DNA/cDNA after amplification using ONT achieves much longer reads and provides more information regarding virome diversity, many of the virome sequences do not have match in current databases. Moreover, direct DNA sequencing of virome provides first insights into the epigenetic modifications on phages, where signals of methylations can be directly detected. Our study demonstrates that progressing sequencing technology and bioinformatic improvements will bring more knowledge into virome composition, diversity and potentially their important functions.</jats:p><jats:sec><jats:title>Highlights</jats:title><jats:list list-type="order"><jats:list-item><jats:p>Virus-like particles were enriched from human stool samples;</jats:p></jats:list-item><jats:list-item><jats:p>Viral nucleotides were sequenced with Oxford Nanopore Technology with and without amplification;</jats:p></jats:list-item><jats:list-item><jats:p>Gut virome in humans showed highly individualized composition;</jats:p></jats:list-item><jats:list-item><jats:p>Novel sequences and contigs were found to be the majority in the resulted sequences;</jats:p></jats:list-item><jats:list-item><jats:p>Epigenetic modifications were detected directly on virus genomes.</jats:p></jats:list-item></jats:list></jats:sec>

Integrated gut virome and bacteriome dynamics in COVID-19 patients

“…Viral DNA and RNA were purified from preparations of enriched virus-like particles (VLPs) obtained from stool samples and characterized by metagenomic sequencing based on methods developed in our lab. 25 As expected, the viromes of COVID-19 patients include DNA/RNA viruses (Herelleviridae family, Virgaviridae family), bacteriophages (Caudovirales order, CrAss-like phage, Inoviridae family, Microviridae family, Myoviridae family, Podoviridae family, and Siphoviridae family) that infect bacterial cells, and some unclassified viruses in the gut ( Figure 2a ). Despite the significant individual differences in viral composition, shared viruses were observed to primarily belong to the Microviridae family.…”

“…Based on the condition of the patients prior to their hospitalization, the disease severity of these subjects was classified into three categories, namely severe (3), moderate (7) and mild (3). Additionally, to compare the dynamics of the bacteriome/virome between healthy people and COVID-19 patients, samples of five healthy individuals from our previous study 25 were selected as healthy controls in this work.…”

“…S6) and five healthy volunteers from our previous study. 25 SARS-CoV-2-infected patients were confirmed using the Novel Coronavirus (SARS-CoV-2) Nucleic Acid Detection kit (BioGerm), as recommended by the CDC in China. According to the Seventh Revised Trial Version of the Novel Coronavirus Pneumonia Diagnosis and Treatment Guidance, 46 the disease severity of patients infected with SARS-CoV-2 was classified into three categories: (1) severe, for adults, respiratory distress (≥ 30 breaths/min), oxygen saturation ≤ 93% at rest and arterial partial pressure of oxygen (PaO 2 )/fraction of inspired oxygen (FiO 2 ) ≤ 300 mmHg (1 mmHg = 0.133 KPa).…”

“…Virus-like particles of inactivated fecal samples were enriched and purified according to the modified workflow we developed in a previous study. 25 In brief, each fecal sample (approximately 500 mg) was resuspended in 15 ml PBS and centrifuged at 4,500 rpm for 10 min at the 4°C to remove large food residues (Beckman Coulter Allegra TM X-22 R). The supernatant was transferred to fresh tubes and centrifuged at 4,500 rpm for 10 min at the 4°C again.…”

Transcription shifts in gut bacteria shared between mothers and their infants: data from the NiPPeR Study

“…However, we did not conduct any viral particle enrichment and cannot therefore easily assess whether any virions of the corresponding phages were present. Sequencing of viral DNA (21,22), in combination with (bulk) metatranscriptomic data, could provide additional insights into abundances of these bacteriophages and their viral life cycles. Additionally, combination of these data would enable investigations of phage-derived lateral gene transfer (of bacterial genes) and their expression (or lack thereof) (23).…”