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.     

Binary Regression Analysis with Pooled Exposure Measurements: A Regression Calibration Approach

Summary It has become increasingly common in epidemiological studies to pool specimens across subjects to achieve accurate quantitation of biomarkers and certain environmental chemicals. In this article, we consider the problem of fitting a binary regression model when an important exposure is subject to pooling. We take a regression calibration approach and derive several methods, including plug‐in methods that use a pooled measurement and other covariate information to predict the exposure level of an individual subject, and normality‐based methods that make further adjustments by assuming normality of calibration errors. Within each class we propose two ways to perform the calibration (covariate augmentation and imputation). These methods are shown in simulation experiments to effectively reduce the bias associated with the naive method that simply substitutes a pooled measurement for all individual measurements in the pool. In particular, the normality‐based imputation method performs reasonably well in a variety of settings, even under skewed distributions of calibration errors. The methods are illustrated using data from the Collaborative Perinatal Project.     

DNA存储中的编码技术

脱氧核糖核酸(Deoxyribonucleic Acid, DNA)是一种天然的信息存储介质,具有存储密度高、存储时间长、损耗率低等特点。在传统存储方式不能满足信息增长的需求时,DNA数据存储技术逐渐成为研究热点。DNA编码是用尽可能少的碱基序列无错的存储数据信息,包括压缩(尽可能少的占用空间)、纠错(无错存储)和转换(数字信息转为碱基序列)3部分。DNA编码是DNA存储中的关键技术,它的结果直接影响存储性能的优劣和数据读写的完整。本文首先介绍DNA存储的发展历史,然后介绍DNA存储的框架,其中重点介绍DNA编码技术,最后对DNA存储中的编解码技术的未来发展方向进行讨论。     

两种生态系统服务价值评估方法比较研究——以四川省金堂县三星镇土地整治工程为例

乡镇土地整治是建设中国美丽乡村的重要途径,对当地生态环境带来正面和负面影响,如何准确评价其生态效益是国家开展土地整治和美丽乡村建设需要面临的重要科学问题。以中国西南部丘陵地区典型乡镇——四川省金堂县三星镇的土地整治工程为例,对比分析了生态系统服务价值当量修正法和单项服务评价法评估土地整治生态效益的异同,以期确定适合我国西南部丘陵地区乡镇土地整治生态效益评估的合理方法。研究结果表明:单项服务评价法的评估结果(生态系统服务价值约300万元)比当量修正法(生态系统服务价值约290万元)高3.5%。当量修正法评估的各项生态系统服务价值增量由高到低依次为:调节服务价值支持服务价值供给服务价值文化服务价值;单项服务评价法评估的生态系统服务价值增量大小排序为:调节服务价值支持服务价值文化服务价值供给服务价值。方法依据和参数的选取不同,导致评估结果具有显著差异,主要表现在单项服务评价法测算的维持养分循环价值、美学景观价值分别是当量修正法的46.90倍和6.94倍,当量修正法测算的水文调节价值是单项服务评价法的5.93倍。单项服务评价法针对各项生态系统服务特征,灵活地选取差异化评估方法,因而其评估结果更准确、真实地反映了生态效益价值。建议运用单项服务评价法评估我国西南部丘陵地区乡镇土地整治的生态效益。     

SIMEX for correction of dietary exposure effects with Box-Cox transformed data

Modelling dietary data, and especially 24-hr dietary recall (24HDR) data, is a challenge. Ignoring the inherent measurement error (ME) leads to biased effect estimates when the association between an exposure and an outcome is investigated. We propose an adapted simulation extrapolation (SIMEX) algorithm for modelling dietary exposures. For this purpose, we exploit the ME model of the NCI method where we assume the assumption of normally distributed errors of the reported intake on the Box-Cox transformed scale and of unbiased recalls on the original scale. According to the SIMEX algorithm, remeasurements of the observed data with additional ME are generated in order to estimate the association between the level of ME and the resulting effect estimate. Subsequently, this association is extrapolated to the case of zero ME to obtain the corrected estimate. We show that the proposed method fulfils the key property of the SIMEX approach, that is, that the MSE of the generated data will converge to zero if the ME variance converges to zero. Furthermore, the method is applied to real 24HDR data of the I.Family study to correct the effects of salt and alcohol intake on blood pressure. In a simulation study, the method is compared with the NCI method resulting in effect estimates with either smaller MSE or smaller bias in certain situations. In addition, we found our method to be more informative and easier to implement. Therefore, we conclude that the proposed method is useful to promote the dissemination of ME correction methods in nutritional epidemiology.     

维甲酸核受体RARα真核表达载体的构建、突变纠正及表达

目的构建维甲酸核受体RARα真核表达载体,并检测其在人肺腺癌细胞A549中表达。方法从小鼠巨噬细胞RAW264.7中提取总RNA,以RT-PCR法扩增RARαcDNA,克隆至真核表达载体pDsRed1-C1中,测序结果显示RARα第1040位A→G,导致其编码蛋白的氨基酸发生改变。通过二次PCR将其纠正,重组载体RedC1-RARα转化大肠埃希菌Top10,筛选阳性克隆做酶切及测序鉴定。脂质体瞬时转染A549细胞,在荧光显微镜下观察RARα的表达。RT-PCR法检测RARα的mRNA水平表达。结果通过RT-PCR及二次PCR得到RARαcDNA,构建其真核表达载体,脂质体瞬时转染A549细胞得到了成功表达,RARα基因产物定位于细胞核内。结论成功构建维甲酸核受体RARα真核表达载体,且证实RARα编码蛋白定位于细胞核内,本研究结果为进一步探讨结核分枝杆菌固有免疫机制奠定了基础。     

北京市湿地生态补偿标准研究

确定合理的湿地生态补偿标准是构建有效湿地生态补偿机制的关键。以北京市境内的湿地为研究对象,利用DPSIR模型明晰北京市湿地生态补偿的机理;基于问卷调查数据,分别利用二元Logistic模型和Tobit模型对影响受调查者受偿态度和受偿意愿的相关因素进行分析;运用条件价值法以及考虑时间价值和经济社会发展阶段的修正湿地生态系统服务价值确定最终的生态补偿标准,并对北京市制定湿地生态补偿机制提出了政策建议。研究结果表明：（1）北京市合理的湿地生态补偿标准为2.728×10⁴-4.84×10⁴元hm^-2a^-1;（2）地区差异、受调查者对湿地保护政策的了解程度以及湿地与社区之间的关系显著影响其受偿态度;（3）地区差异、年龄、受教育程度、家庭收入情况、对生态补偿的了解程度显著影响其受偿意愿;（4）为减少将来推进湿地生态补偿过程中可能遭遇的潜在阻碍,北京市应该加强湿地保护相关政策的宣传普及工作,注重对基层政策落实情况的监督,协调湿地政策与社区的关系;（5）有必要结合地区特征制定差异化的湿地生态补偿标准。研究结果可为今后北京市探索建立湿地生态补偿机制、提升湿地保护效率提供科学数据和理论参考。     

A possible mechanism for the dynamics of transition between polymerase and exonuclease sites in a high-fidelity DNA polymerase

The fidelity of DNA synthesis by DNA polymerase is significantly increased by a mechanism of proofreading that is performed at the exonuclease active site separate from the polymerase active site. Thus, the transition of DNA between the two active sites is an important activity of DNA polymerase. Here, based on our proposed model, the rates of DNA transition between the two active sites are theoretically studied. With the relevant parameters, which are determined from the available crystal structure and other experimental data, the calculated transfer rate of correctly base-paired DNA from the polymerase to exonuclease sites and the transfer rate after incorporation of a mismatched base are in good agreement with the available experimental data. The transfer rates in the presence of two and three mismatched bases are also consistent with the previous experimental data. In addition, the calculated transfer rate from the exonuclease to polymerase sites has a large value even with the high binding affinity of 3′-5′ ssDNA for the exonuclease site, which is also consistent with the available experimental value. Moreover, we also give some predictive results for the transfer rate of DNA containing only A:T base pairs and that of DNA containing only G:C base pairs.