PDF(Pubmed)
Abstract：
Routinely used metagenomic next-generation sequencing (mNGS) techniques often fail to detect low-level viremia (<104 copies/mL) and appear biased towards viruses with linear genomes. These limitations hinder the capacity to comprehensively characterize viral infections, such as those attributed to the Anelloviridae family. These near ubiquitous non-pathogenic components of the human virome have circular single-stranded DNA genomes that vary in size from 2.0 to 3.9 kb and exhibit high genetic diversity. Hence, species identification using short reads can be challenging. Here, we introduce a rolling circle amplification (RCA)-based metagenomic sequencing protocol tailored for circular single-stranded DNA genomes, utilizing the long-read Oxford Nanopore platform. The approach was assessed by sequencing anelloviruses in plasma drawn from people who inject drugs (PWID) in two geographically distinct cohorts. We detail the methodological adjustments implemented to overcome difficulties inherent in sequencing circular genomes and describe a computational pipeline focused on anellovirus detection. We assessed our protocol across various sample dilutions and successfully differentiated anellovirus sequences in conditions simulating mixed infections. This method provides a robust framework for the comprehensive characterization of circular viruses within the human virome using the Oxford Nanopore.
摘要：
常规使用的宏基因组下一代测序（mNGS）技术通常无法检测到低水平病毒血症（<104拷贝/mL），并且似乎偏向于具有线性基因组的病毒。这些限制阻碍了全面表征病毒感染的能力，如那些归于无带病毒科的。人类病毒的这些几乎普遍存在的非致病性成分具有环状单链DNA基因组，其大小从2.0kb到3.9kb不等，并表现出高度的遗传多样性。因此，使用短读数进行物种鉴定可能是具有挑战性的。这里,我们介绍了一种基于滚环扩增（RCA）的宏基因组测序方案，该方案适用于环状单链DNA基因组，利用长期阅读的牛津纳米孔平台。通过对从两个地理上不同的队列中的注射药物（PWID）的人中提取的血浆中的anellovirus进行测序来评估该方法。我们详细介绍了为克服循环基因组测序固有困难而进行的方法学调整，并描述了专注于anellovirus检测的计算流程。我们在各种样品稀释度评估了我们的方案，并在模拟混合感染的条件下成功区分了anellovirus序列。该方法为使用牛津纳米孔全面表征人类病毒体内的环状病毒提供了强大的框架。
