Distinguishing low frequency mutations from RT-PCR and sequence errors in viral deep sequencing data

Background

RNA viruses have high mutation rates and exist within their hosts as large, complex and heterogeneous populations, comprising a spectrum of related but non-identical genome sequences. Next generation sequencing is revolutionising the study of viral populations by enabling the ultra deep sequencing of their genomes, and the subsequent identification of the full spectrum of variants within the population. Identification of low frequency variants is important for our understanding of mutational dynamics, disease progression, immune pressure, and for the detection of drug resistant or pathogenic mutations. However, the current challenge is to accurately model the errors in the sequence data and distinguish real viral variants, particularly those that exist at low frequency, from errors introduced during sequencing and sample processing, which can both be substantial.

Results

We have created a novel set of laboratory control samples that are derived from a plasmid containing a full-length viral genome with extremely limited diversity in the starting population. One sample was sequenced without PCR amplification whilst the other samples were subjected to increasing amounts of RT and PCR amplification prior to ultra-deep sequencing. This enabled the level of error introduced by the RT and PCR processes to be assessed and minimum frequency thresholds to be set for true viral variant identification. We developed a genome-scale computational model of the sample processing and NGS calling process to gain a detailed understanding of the errors at each step, which predicted that RT and PCR errors are more likely to occur at some genomic sites than others. The model can also be used to investigate whether the number of observed mutations at a given site of interest is greater than would be expected from processing errors alone in any NGS data set. After providing basic sample processing information and the site’s coverage and quality scores, the model utilises the fitted RT-PCR error distributions to simulate the number of mutations that would be observed from processing errors alone.

Conclusions

These data sets and models provide an effective means of separating true viral mutations from those erroneously introduced during sample processing and sequencing.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1456-x) contains supplementary material, which is available to authorized users.     

A Multicenter Study of Viral Aetiology of Community-Acquired Pneumonia in Hospitalized Children in Chinese Mainland

Virologica Sinica - Community-acquired pneumonia (CAP) is one of the leading causes of morbidity and mortality in children worldwide. In this study, we aimed to describe the aetiology of viral...     

抗生素诱导的呼吸道菌群缺失对小鼠RSV感染的影响

目的 探究抗生素雾化暴露引起的呼吸道菌群缺失对小鼠呼吸道合胞病毒(RSV)感染的影响,为临床合理使用抗生素提供指导意见。 方法 32只BALB/c小鼠分为2组:雾化ddH2O对照组和雾化ABX组合抗生素组,处理6 d后,进行细菌16S rRNA基因PCR检测,构建呼吸道菌群缺失小鼠模型。上述2组组内再随机分为2小组,即PBS对照组(ddH2O+PBS,ABX+PBS)和RSV感染组(ddH2O+RSV,ABX+RSV),饲养至第14天。检测和分析各组小鼠支气管肺泡灌洗液(BALF)中的炎症细胞和相关细胞因子(TNFα、IL8、IL10及MCP1)的数量和水平,观察肺组织病理学状况及检测病毒载量。 结果 BALF中细菌DNA提取和16S rRNA基因PCR检测显示,雾化ABX组合抗生素处理能够有效地剔除呼吸道菌群。BALF中炎症细胞和相关细胞因子检测显示,ABX+RSV组炎症细胞总数明显增多(P2O+RSV组和ABX+RSV组小鼠肺部损伤明显加重(均P≤0.01),与ddH2O+RSV组相比较,ABX+RSV组的病毒载量明显升高(t=2.716 0,P=0.021 7)。 结论 雾化ABX组合抗生素不仅能够有效地剔除呼吸道菌群,而且明显增加了小鼠感染RSV的风险,导致呼吸道炎症加重,以及病毒载量升高。     

Human herpesvirus-8-encoded signalling ligands and receptors

Analysis of the genome of human herpesvirus 8 (HHV-8) led to the discovery of several novel genes, unique among the characterized gammaherpesviruses. These include cytokines (interleukin-6 and chemokine homologues), two putative signal-transducing transmembrane proteins encoded by genes K1 and K15 at the genome termini, and an OX-2 (CD200) receptor homologue that had not previously been identified in a gammaherpesvirus. HHV-8 also specifies a diverged version of the gammaherpesvirus-conserved G protein-coupled chemokine receptor (vGCR) and a latently expressed protein unique to HHV-8 specified by open reading frame (ORF) K12. These cytokine and receptor homologues mediate signal transduction or modulate the activities of other endogenous cytokines and receptors to enhance viral productive replication, regulate latent-lytic switching, evade host attack, or mediate cell survival. The viral signalling ligands and receptors are also potential contributors to virus-associated diseases, Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease, and so represent potentially important targets for therapeutic and antiviral drugs. Understanding these proteins' modes of action and functions in viral biology and disease is therefore of considerable importance, and the subject of this review.     

Viral metagenomics analysis of planktonic viruses in East Lake, Wuhan, China

East Lake (Lake Donghu), located in Wuhan, China, is a typical city freshwater lake that has been experiencing eutrophic conditions and algal blooming during recent years. Marine and fresh water are considered to contain a large number of viruses. However, little is known about their genetic diversity because of the limited techniques for culturing viruses. In this study, we conducted a viral metagenomic analysis using a high-throughput sequencing technique with samples collected from East Lake in Spring, Summer, Autumn, and Winter. The libraries from four samples each generated 234,669, 71,837, 12,820, and 34,236 contigs (> 90 bp each), respectively. The genetic structure of the viral community revealed a high genetic diversity covering 23 viral families, with the majority of contigs homologous to DNA viruses, including members of Myoviridae, Podoviridae, Siphoviridae, Phycodnaviridae, and Microviridae, which infect bacteria or algae, and members of Circoviridae, which infect invertebrates and vertebrates. The highest viral genetic diversity occurred in samples collected in August, then December and June, and the least diversity in March. Most contigs have low-sequence identities with known viruses. PCR detection targeting the conserved sequences of genes (g20, psbA, psbD, and DNApol) of cyanophages further confirmed that there are novel cyanophages in the East Lake. Our viral metagenomic data provide the first preliminary understanding of the virome in one freshwater lake in China and would be helpful for novel virus discovery and the control of algal blooming in the future.     

Gamma irradiation of animal sera for inactivation of viruses and mollicutes – A review

Animal-derived materials such as animal sera represent a low, but finite, risk for introduction of an adventitious agent (virus or mollicute) into a biological bulk harvest during upstream manufacturing processes involving mammalian cell substrates. Viral and mollicute (Mycoplasma sp. and Acholeplasma sp.) contamination events have been relatively rare, but many of those that have been reported have been attributed to use of infected animal sera in growth media during cell expansion. The risk of introduction of viruses and mollicutes may be mitigated by elimination of the use of animal sera and implementation instead of chemically defined or serum- and animal-derived material-free cell culture media. When use of animal sera is unavoidable, however, mitigation of the risk of introducing an adventitious contaminant may involve treatment of the sera to inactivate potential contaminants. Gamma irradiation is one of the most widely employed methods for viral and mollicute inactivation in animal sera. In this article, we review the inactivation results reported for viral and mollicute inactivation in frozen serum. Studies performed to assess the impact of gamma irradiation on serum quality and performance are also discussed. The available data indicate that inactivation of mollicutes in serum is essentially complete at the gamma radiation doses normally employed (25–40 kGy), while the efficacy and kinetics for viral inactivation in serum by gamma irradiation appear to be dependent in part upon the size of the target virus.     

婴幼儿病毒性脑炎并发神经源性肺水肿血糖及电解质检测的意义

目的探讨婴幼儿病毒性脑炎并发神经源性肺水肿(neurogenic pulmonary edema,NPE)血糖、血清电解质的变化及与病情发展的关系。方法选择2008年6月至2010年11月蚌埠医学院第一附属医院收治的婴幼儿病毒性脑炎患儿19例为研究对象,依据患儿是否并发NPE分为2组,NPE组9例,非NPE组10例。以入院后第1次静脉血的血糖和电解质为评价标准,并应用t检验和四格表资料的Fisher确切概率法,进行血糖和血清电解质比较。结果 NPE组患儿血糖水平[(19.24±9.64)mmol/L]显著高于非NPE组[(4.90±1.11)mmol/L](t=4.44,P<0.01),而血钙水平[(1.75±0.32)mmol/L]显著低于非NPE组[(2.37±0.17)mmol/L](t=-5.31,P<0.01)。NPE组高血糖和低钙血症发生率远大于非NPE组(P<0.01)。2组血清钾、钠、氯变化差异无统计学意义(P>0.05)。结论婴幼儿病毒性脑炎并发NPE可引起血糖改变和电解质紊乱。高血糖和低钙血症是导致NPE的危险因素,早期检测血糖及血钙水平可作为判断婴幼儿病毒性脑炎并发NPE病情和预后的有效指标。     

Evaluation of the role of the vaccinia virus uracil DNA glycosylase and A20 proteins as intrinsic components of the DNA polymerase holoenzyme

The vaccinia virus DNA polymerase is inherently distributive but acquires processivity by associating with a heterodimeric processivity factor comprised of the viral A20 and D4 proteins. D4 is also an enzymatically active uracil DNA glycosylase (UDG). The presence of an active repair protein as an essential component of the polymerase holoenzyme is a unique feature of the replication machinery. We have shown previously that the A20-UDG complex has a stoichiometry of ～1:1, and our data suggest that A20 serves as a bridge between polymerase and UDG. Here we show that conserved hydrophobic residues in the N' terminus of A20 are important for its binding to UDG. Our data argue against the assembly of D4 into higher order multimers, suggesting that the processivity factor does not form a toroidal ring around the DNA. Instead, we hypothesize that the intrinsic, processive DNA scanning activity of UDG tethers the holoenzyme to the DNA template. The inclusion of UDG as an essential holoenzyme component suggests that replication and base excision repair may be coupled. Here we show that the DNA polymerase can utilize dUTP as a substrate in vitro. Moreover, uracil moieties incorporated into the nascent strand during holoenzyme-mediated DNA synthesis can be excised by the viral UDG present within this holoenzyme, leaving abasic sites. Finally, we show that the polymerase stalls upon encountering an abasic site in the template strand, indicating that, like many replicative polymerases, the poxviral holoenzyme cannot perform translesion synthesis across an abasic site.     

Feeding behaviour and performance of different populations of the black currant‐lettuce aphid,Nasonovia ribisnigri,on resistant and susceptible lettuce

When crops are bred for resistance to herbivores, these herbivores are under strong selection pressure to overcome this resistance, which may result in the emergence of virulent biotypes. This is a growing problem for crop species attacked by aphids. The Nr‐gene in lettuce confers near‐complete resistance against the black currant‐lettuce aphid, Nasonovia ribisnigri (Mosely) (Hemiptera: Aphididae). Since 2007, populations of N. ribisnigri have been reported in several locations in Europe to infest resistant lettuce varieties that possess the Nr‐gene. The objective of this study was to analyse the behaviour and level of virulence of several N. ribisnigri populations observed to have colonized Nr‐locus‐containing lettuce lines. We analysed the stylet penetration and feeding behaviour, and the performance of these N. ribisnigri populations on resistant and susceptible lettuce lines. Large variation in the degree of virulence to the Nr‐locus‐containing lettuce lines was found among populations of the Nr:1 biotype. The German population was highly virulent on the Nr‐containing resistant lettuce lines, and showed similar feeding behaviour and performance on both the susceptible and resistant lettuces. The French population from Paris was the second most virulent, though reproduction on the resistant lines was reduced. The French population from Perpignan and a population from Belgium, however, showed reduced performance and feeding rate on the resistant compared to the susceptible lettuces. The lettuce background in which the Nr‐gene is expressed influences the level of resistance to the various Nr:1 aphid populations, because the performance and feeding behaviour differed between the aphids on the cultivars (romaine lettuce) compared to the near‐isogenic lines (butterhead/iceberg lettuce). This study also shows that being able to feed on a plant not automatically implies that a population can successfully develop on that plant, because aphids showed phloem ingestion during the 8‐h recording period on resistant lettuce, but were not able to survive and reproduce on the same lettuce line.     

Highly Divergent T-cell Receptor Binding Modes Underlie Specific Recognition of a Bulged Viral Peptide bound to a Human Leukocyte Antigen Class I Molecule

Human leukocyte antigen (HLA)-I molecules can present long peptides, yet the mechanisms by which T-cell receptors (TCRs) recognize featured pHLA-I landscapes are unclear. We compared the binding modes of three distinct human TCRs, CA5, SB27, and SB47, complexed with a “super-bulged” viral peptide (LPEPLPQGQLTAY) restricted by HLA-B*35:08. The CA5 and SB27 TCRs engaged HLA-B*35:08^LPEP similarly, straddling the central region of the peptide but making limited contacts with HLA-B*35:08. Remarkably, the CA5 TCR did not contact the α1-helix of HLA-B*35:08. Differences in the CDR3β loop between the CA5 and SB27 TCRs caused altered fine specificities. Surprisingly, the SB47 TCR engaged HLA-B*35:08^LPEP using a completely distinct binding mechanism, namely “bypassing” the bulged peptide and making extensive contacts with the extreme N-terminal end of HLA-B*35:08. This docking footprint included HLA-I residues not observed previously as TCR contact sites. The three TCRs exhibited differing patterns of alloreactivity toward closely related or distinct HLA-I allotypes. Thus, the human T-cell repertoire comprises a range of TCRs that can interact with “bulged” pHLA-I epitopes using unpredictable strategies, including the adoption of atypical footprints on the MHC-I.