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Ultradeep Pyrosequencing of Hepatitis C Virus to Define Evolutionary Phenotypes
超深度焦磷酸测序鉴定丙型肝炎病毒的进化表型   

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Abstract

Analysis of hypervariable regions (HVR) using pyrosequencing techniques is hampered by the ability of error correction algorithms to account for the heterogeneity of the variants present. Analysis of between-sample fluctuations to virome sub-populations, and detection of low frequency variants, are unreliable through the application of arbitrary frequency cut offs. Cumulatively this leads to an underestimation of genetic diversity. In the following technique we describe the analysis of Hepatitis C virus (HCV) HVR1 which includes the E1/E2 glycoprotein gene junction. This procedure describes the evolution of HCV in a treatment naïve environment, from 10 samples collected over 10 years, using ultradeep pyrosequencing (UDPS) performed on the Roche GS FLX titanium platform (Palmer et al., 2014). Initial clonal analysis of serum samples was used to inform downstream error correction algorithms that allowed for a greater sequence depth to be reached. PCR amplification of this region has been tested for HCV genotypes 1, 2, 3 and 4.

Keywords: Ultradeep pyrosequencing(超深度焦磷酸测序), Virus(病毒), Quasispecies(准种), Hypervariability(高变性)

Background

Analysis of UDPS datasets derived from virus amplicons frequently relies on software tools that are not optimized for amplicon analysis, assume random incorporation of sequencing mutations and are focused on finding true sequences rather than false variants. These difficulties are further complicated by the presence of hypervariable regions present in RNA virus genomes. Many studies utilizing UDPS look to overcome these issues by applying arbitrary frequency cut offs to the data, resulting in the loss of minor variants. Here, a temporally matched clonal dataset, together with an error correction methodology designed to overcome the problems outlined, facilitated the retention of valuable sequence information.

Materials and Reagents

  1. 1.5 ml tube (SARSTEDT, catalog number: 72.690.001 )
  2. 200 µl MicroAmp® PCR tube (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: N8010840 )
  3. Clean stainless steel blade
  4. One Shot® TOP10 Competent Cells (Thermo Fisher Scientific, InvitrogenTM, catalog number: C404003 )
  5. QIAamp® Viral RNA mini kit (QIAGEN, catalog number: 52904 )
  6. Random primer (Promega, catalog number: C1181 )
  7. Deoxynucleoside triphosphate (dNTP’s, 100 mM) set, PCR grade (Roche Molecular Systems, catalog number: 11969064001 )
  8. AMV reverse transcriptase (Promega, catalog number: M5101 )
  9. RNasin® Ribonuclease inhibitor (Promega, catalog number: N2511 )
  10. Outer-forward primer: 5’- ATGGCATGGGATATGAT -3’ (10 pmol/µl, Eurofins)
  11. Outer-reverse primer: 5’- AAGGCCGTCCTGTTGA -3’ (10 pmol/µl, Eurofins)
  12. Inner-forward primer: 5’- GCATGGGATATGATGATGAA -3’ (10 pmol/µl, Eurofins)
  13. Inner-reverse primer: 5’- GTCCTGTTGATGTGCCA -3’ (10 pmol/µl, Eurofins)
  14. Pwo DNA polymerase (5 U/µl,) including 10x reaction buffer (- MgSO4) and MgSO4 stock solution (25 mM) (Roche Molecular Systems, catalog number: 11644955001 )
  15. dH2O (Sigma-Aldrich, catalog number: W4502 )
  16. Sybr safe DNA gel stain (Thermo Fisher Scientific, InvitrogenTM, catalog number: S33102 )
  17. Agarose (Sigma-Aldrich, catalog number: A9539 )
  18. GeneRuler 100 bp Plus DNA ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SM0323 )
  19. Gel extraction kit (QIAGEN, catalog number: 28704 )
  20. CloneJet PCR Cloning Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: K1231 )
  21. GeneJet Plasmid Miniprep Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: K0503 )
  22. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  23. Acetic acid glacial (BDH Laboratory Supplies, catalog number: 10001CU )
  24. Ethylenediaminetetraacetic acid solution 0.5 M (EDTA) (Sigma-Aldrich, catalog number: 03690 )
  25. 1x TAE (see Recipes)

Equipment

  1. PCR thermal cycler (Thermo Fisher Scientific, Applied BiosystemsTM, model: Applied Biosystems® 2720 )
  2. BioPhotometer (Eppendorf, http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf)
  3. Water bath (JULABO, model: SW22 )
  4. Orbital shaker incubator (Grant, model: ES-80 )
  5. Ultraviolet transilluminator (UVP, model: TMW-20 )

Software

  1. SFFFile tools (Roche Molecular Systems)
  2. k-mer error correction (KEC) and empirical threshold (ET) (Skums et al., 2012)
  3. MEGA 6.0 (Tamura et al., 2013)

Procedure

  1. RNA extraction and cDNA generation
    1. Whole patient serum, surplus to diagnostic testing requirements and with a mean viral titer of 6 HCV RNA log10 IU/ml, was used as the starting material.
    2. RNA was extracted from 140 µl of serum using QIAamp® Viral RNA mini kit according to the manufacturer’s instructions into 1.5 ml RNase free tubes and a final volume of 50 µl.
    3. 11 µl of extracted viral RNA was mixed with 1 µl (0.5 µg) random primer.
    4. Samples were incubated at 75 °C for 10 min.
    5. To this was added a master mix which contained 2 µl (80 mM) dNTP mix, 1 µl (10 U) AMV reverse transcriptase, 1 µl (40 U) RNasin, 4 µl AMV reaction buffer.
    6. cDNA generation took place at 42 °C for 60 min, followed by 94 °C for 3 min.
    7. Samples were kept at 4 °C until required.

  2. Nested PCR to amplify the HCV E1/E2 gene junction
    1. Prepare the primary PCR master mix to a final volume of 45 µl:
      Outer-forward primer:
      1.5 µl
      Outer-reverse primer:
      1.5 µl
      10x reaction buffer (- MgSO4):
      5 µl
      dNTP mix:
      1 µl
      MgSO4 stock solution: 
      3 µl
      Pwo: 
      0.5 µl
      PCR grade water:
      32.5 µl
    2. 5 µl of cDNA is then added to the master mix.
    3. 1° PCR cycle parameters:
      1. Initial denaturation: 3 min at 94 °C
      2. Cycle conditions (repeat for 35 cycles):
        Denaturation: 15 sec at 94 °C
        Annealing: 30 sec at 51 °C
        Extension: 30 sec at 72 °C
      3. Final extension: 7 min at 72 °C
    4. Keep sample at 4 °C until required.
    5. Prepare master mix for secondary PCR to a final volume of 46 µl:
      Inner-forward primer:
      1.5 µl
      Inner-reverse primer:
      1.5 µl
      10x reaction buffer (- MgSO4):
      5 µl
      dNTP mix:
      1 µl
      MgSO4 stock solution:
      2 µl
      Pwo: 
      0.5 µl
      PCR grade water: 
      34.5 µl
    6. 4 µl of primary PCR sample is then added to the master mix.
    7. 2° PCR cycle parameters:
      1. Initial denaturation: 3 min at 94 °C
      2. Cycle conditions (repeat for 35 cycles):
        Denaturation: 15 sec at 94 °C
        Annealing: 30 sec at 53 °C
        Extension: 30 sec at 72 °C
      3. Final extension: 7 min at 72 °C
    8. Samples were kept at 4 °C until required.
    9. To ensure that the initial amount of the template was not limiting, 1:100 dilution of the viral RNA was prepared which, when used as the starting template for nested PCR as described, should yield an amplicon visualized by gel electrophoresis for each sample.

  3. Preparation of samples for pyrosequencing
    1. Two 2% TAE agarose gels were poured, one containing Sybr safe DNA gel stain and one without.
    2. Once set, the gels were split in two, with one half of the gel containing the gel stain joined with the second gel without gel stain.
    3. The 50 µl amplicon sample was split in two (10 µl and 40 µl) and resolved on the above gel. The 10 µl sample was stained using Sybr safe, while the 40 µl sample was not stained and went forward for downstream procedures. The resultant amplicon in this instance was 320 bp (Figure 1).
    4. The region of the gel containing the unstained band (40 µl sample) was cut out using a clean stainless steel blade using the stained 10 µl sample as a positioning guide and transferred to a clean 1.5 ml tube.
    5. The amplicon was gel extracted using a gel extraction kit according to the manufacturer’s instructions.
    6. Extracted amplicons were quantified using a BioPhotometer.
    7. Samples were prepared in equimolar concentrations and diluted to a final concentration of 1 x 107 molecules/ml.
    8. Pyrosequencing was outsourced to Roche 454 Life Sciences (Brandford, CT, USA).


      Figure 1. Amplicon visualization. Successful amplification of the 320 bp amplicon was confirmed following agarose gel electrophoresis. 10 µl of the 2° PCR sample was loaded.

  4. Clonal analysis
    1. Purified amplicons were cloned using CloneJet PCR Cloning Kit and transformed into One Shot® TOP10 Competent Cells using the manufacturer’s instructions using a molar ratio of 3:1 insert to vector.
    2. 20 clones per sample were generated.
    3. Plasmids were purified using GeneJet Plasmid Miniprep Kit as per manufacturer’s instructions.
    4. Sequencing of E1/E2 inserts was performed by Eurofins.
    5. All trace files were inspected to exclude sequences where double peaks or regions of ambiguous sequence were present.

  5. Data handling and error correction
    1. The raw sff data files were managed using SFFFile tools.
    2.  Low-quality reads and reads shorter than 90% of the expected amplicon lengths were removed.
    3. Phylogenetic separation of the clonal data using a general time-reversible model with gamma-distributed and invariant sites (GTR+G+I) using MEGA 6.0 (Tamura et al., 2013).
    4.  Main branches with bootstrap values (of 1,000 resamplings) > 85 were categorised as (sub-)lineages (Palmer et al., 2014).
    5. Two 24-bp motifs, that defined the HVR1 amino acid profile of each (sub-)lineage, were subsequently applied to the sequence analysis pipeline. The first 15-bp of the motif span the conserved 3’-end of E1. The remaining 9-bp include the first three amino acids of the HVR1 at the 5’-end of E2.
    6. The overall number of motifs used reflected the observed changes in the dominant HVR1 over time. For each (sub-)lineage, two motif reference sequences were deemed sufficient.
    7. To increase the sensitivity of the sequencing error correction algorithms (KEC-ET), the UDPS data was partitioned according to the presence of corresponding motifs.
    8. In order to ensure the quality of the analyzed data and the absence of PCR and sequencing chimeras, reads that had more than a 3 bp difference from the best-matching sequence from this motif set were removed.
    9. KEC consists of the three stages
      1. In stage 1, the set of k-mers (substring of fixed length k) of reads from the processed data set is calculated and the distribution of frequencies of k-mers is analyzed. The error threshold is calculated as the minimal frequency of k-mers separating two different distributions.
      2. In stage 2, k-mers with frequencies lower than the error threshold are considered erroneous and are used to identify and correct the errors. The corrections are based on an analysis of different factors, including the length of a segment of consecutive erroneous k-mers, the sequences of nucleotides at the end of that segment, and the frequencies of the similar correct k-mers. The procedure of error correction is repeated iteratively i times.
      3. In stage 3, the reads containing k-mers that were not corrected in stage 2 are discarded.
    10. The following parameters of KEC were used: k = 25 and i = 3.

Data analysis

A more complete description of the data handling and error correction procedure can be found in the original article, http://jvi.asm.org/content/88/23/13709.short (Palmer et al., 2014).

Notes

All serum samples were genotyped and quantified by the Molecular Virology Diagnostic & Research Laboratory at Cork University Hospital, Cork, Ireland. https://www.ucc.ie/en/meddept/people/liam-fanning/mvdrl/

Recipes

  1. 1x TAE
    4.84 g Tris base
    1.15 ml acetic acid glacial
    2 ml 0.5 M EDTA
    Add dH2O to 1 L

References

  1. Palmer, B. A., Dimitrova, Z., Skums, P., Crosbie, O., Kenny-Walsh, E. and Fanning, L. J. (2014). Analysis of the evolution and structure of a complex intrahost viral population in chronic hepatitis C virus mapped by ultradeep pyrosequencing. J Virol 88(23): 13709-13721.
  2. Skums, P., Dimitrova, Z., Campo, D. S., Vaughan, G., Rossi, L., Forbi, J. C., Yokosawa, J., Zelikovsky, A. and Khudyakov, Y. (2012). Efficient error correction for next-generation sequencing of viral amplicons. BMC Bioinformatics 13 Suppl 10: S6.
  3. Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30(12): 2725-2729.

简介

使用焦磷酸测序技术的高变区(HVR)分析受到纠错算法解释存在的变异异质性的能力的阻碍。通过应用任意频率切断,对样本间波动与色情子群体的分析以及低频变体的检测是不可靠的。累积地导致遗传多样性的低估。在以下技术中,我们描述了包含E1 / E2糖蛋白基因连接的丙型肝炎病毒(HCV)HVR1的分析。该程序描述了HCV在治疗初始环境中的演变,从10年来收集的10个样品中,使用在Roche GS FLX钛平台上进行的超深度焦磷酸测序(UDPS)(2014年,Palmer等人) 。使用血清样品的初步克隆分析来通知允许达到更大序列深度的下游误差校正算法。已经针对HCV基因型1,2,3和4测试了该区域的PCR扩增。

背景 衍生自病毒扩增子的UDPS数据集的分析经常依赖于未针对扩增子分析进行优化的软件工具,假设随机并入测序突变,并且集中在找到真实序列而不是假变异体。存在于RNA病毒基因组中的高变区存在这些困难。许多利用UDPS的研究通过对数据应用任意的频率切断来寻求解决这些问题,从而导致小的变体的丢失。在这里,暂时匹配的克隆数据集以及旨在克服所概述的问题的纠错方法,有助于保留有价值的序列信息。

关键字:超深度焦磷酸测序, 病毒, 准种, 高变性

材料和试剂

  1. 1.5ml管(SARSTEDT,目录号:72.690.001)
  2. 将200μLMicroAmp PCR(Thermo Fisher Scientific,Applied Biosystems TM,目录号:N8010840)PCR管
  3. 清洁不锈钢刀片
  4. One Shot ® TOP10感受态细胞(Thermo Fisher Scientific,Invitrogen TM,目录号:C404003)
  5. QIAamp ®病毒RNA迷你试剂盒(QIAGEN,目录号:52904)
  6. 随机引物(Promega,目录号:C1181)
  7. 脱氧核苷三磷酸(dNTP's,100mM),PCR等级(Roche Molecular Systems,目录号:11969064001)
  8. AMV逆转录酶(Promega,目录号:M5101)
  9. RNasin ®核糖核酸酶抑制剂(Promega,目录号:N2511)
  10. 外向引物:5'-ATGGCATGGGATATGAT-3'(10pmol /μl,Eurofins)
  11. 外反向引物:5'-AAGGCCGTCCTGTTGA -3'(10pmol /μl,Eurofins)
  12. 内转基因引物:5'-GCATGGGATATGATGATGAA -3'(10pmol /μl,Eurofins)
  13. 内反向引物:5'-GTCCTGTTGATGTGCCA -3'(10pmol /μl,Eurofins)
  14. 包含10x反应缓冲液( - MgSO 4)和MgSO 4的储备溶液(25mM)的Pwo DNA聚合酶(5U /μl)(Roche Molecular Systems,目录号: 11644955001)
  15. dH 2 O(Sigma-Aldrich,目录号:W4502)
  16. Sybr安全DNA凝胶染色(Thermo Fisher Scientific,Invitrogen TM,目录号:S33102)
  17. 琼脂糖(Sigma-Aldrich,目录号:A9539)
  18. GeneRuler 100 bp Plus DNA梯(Thermo Fisher Scientific,Thermo Scientific TM,目录号:SM0323)
  19. 凝胶提取试剂盒(QIAGEN,目录号:28704)
  20. CloneJet PCR克隆试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,目录号:K1231)
  21. GeneJet Plasmid Miniprep Kit(Thermo Fisher Scientific,Thermo Scientific TM,目录号:K0503)
  22. Trizma ®基质(Sigma-Aldrich,目录号:T1503)
  23. 乙酸冰川(BDH实验室用品,目录号:10001CU)
  24. 乙二胺四乙酸溶液0.5M(EDTA)(Sigma-Aldrich,目录号:03690)
  25. 1x TAE(见食谱)

设备

  1. PCR热循环仪(Thermo Fisher Scientific,Applied Biosystems TM,型号:Applied Biosystems 2720)
  2. BioPhotometer(Eppendorf, http://植物园。 harvard.edu/wp-content/uploads/Biophotometer-manual.pdf
  3. 水浴(JULABO,型号:SW22)
  4. 轨道摇床培养箱(Grant,型号:ES-80)
  5. 紫外透射仪(UVP,型号:TMW-20)

软件

  1. SFFFile工具(Roche Molecular Systems)
  2. k-mer误差校正(KEC)和经验阈值(ET)(Skums等人,2012)
  3. MEGA 6.0(Tamura等,,2013)

程序

  1. RNA提取和cDNA生成
    1. 全身患者血清,多余至诊断检测要求,平均病毒滴度为6 HCV RNA log 10μl/ml,用作起始物质。
    2. 使用QIAamp 病毒RNA迷你试剂盒,根据制造商的说明书,从140μl血清中提取RNA,将其放入1.5ml无RNase的试管中,最终体积为50μl。
    3. 将11μl提取的病毒RNA与1μl(0.5μg)随机引物混合
    4. 样品在75℃下孵育10分钟
    5. 向其中加入含有2μl(80mM)dNTP混合物,1μl(10U)AMV逆转录酶,1μl(40U)RNasin,4μlAMV反应缓冲液的母液混合物。
    6. cDNA在42℃进行60分钟,94℃3分钟
    7. 样品保存在4℃,直到需要
  2. 嵌套PCR扩增HCV E1/E2基因连接
    1. 准备初级PCR主混合物,最终体积为45μl:
      外转底稿:
      1.5μl
      外反向引物:
      1.5μl
      10x反应缓冲液( - MgSO 4) 4 ):
      5μl
      dNTP组合:
      1μl
      硫酸盐 4 库存解决方案: 
      3μl
      Pwo: 
      0.5μl
      PCR级水:
      32.5微升
    2. 然后将5μlcDNA加入到主混合物中。
    3. 1°PCR循环参数:
      1. 初始变性:94℃3分钟
      2. 周期条件(重复35个周期):
        变性:在94℃下15秒
        退火:51°C 30秒
        延长:72°C 30秒
      3. 最终延伸:72°C 7分钟
    4. 将样品保持在4°C直至需要。
    5. 准备二级PCR的主混合物,最终体积为46μl:
      内向前引物:
      1.5μl
      内反向引物:
      1.5μl
      10x反应缓冲液( - MgSO 4) 4 ):
      5μl
      dNTP组合:
      1μl
      硫酸盐 4 库存解决方案:
      2μl
      Pwo: 
      0.5μl
      PCR级水: 
      34.5微升
    6. 然后将4μl初级PCR样品加入到主混合物中。
    7. 2°PCR循环参数:
      1. 初始变性:94℃3分钟
      2. 周期条件(重复35个周期):
        变性:在94℃下15秒
        退火:53°C 30秒
        延长:72°C 30秒
      3. 最终延伸:72°C 7分钟
    8. 样品保存在4℃,直到需要
    9. 为了确保模板的初始量不受限制,制备1:100稀释的病毒RNA,当用作所述的巢式PCR的起始模板时,应产生通过凝胶电泳显示每个样品的扩增子。 br />
  3. 制备焦磷酸测序样品
    1. 倒入2个2%的TAE琼脂糖凝胶,一个含有Sybr安全DNA凝胶染色剂,一个不含
    2. 一旦凝固,凝胶分成两部分,其中一半的凝胶含凝胶染色剂与第二个凝胶结合而没有凝胶染色。
    3. 将50μl扩增子样品分成两个(10μl和40μl),并在上述凝胶上分离。使用Sybr safe将10μl样品染色,而40μl样品未染色并向下进行下游。在这种情况下,所得的扩增子为320bp(图1)
    4. 使用干净的不锈钢刀片将含有未染色带(40μl样品)的凝胶区域切割,使用染色的10μl样品作为定位引导物,并转移到干净的1.5ml管中。
    5. 使用凝胶提取试剂盒根据制造商的说明书将扩增子凝胶提取。
    6. 使用BioPhotometer量化提取的扩增子。
    7. 以等摩尔浓度制备样品,稀释至终浓度为1×10 7分子/ml。
    8. 焦磷酸测序外包给Roche 454生命科学(Brandford,CT,USA)。


      图1扩增子可视化在琼脂糖凝胶电泳之后证实了320bp扩增子的成功扩增。加载10μl2°PCR样品。

  4. 克隆分析
    1. 使用CloneJet PCR克隆试剂盒克隆纯化的扩增子,并使用制造商的说明书,使用3:1插入物与载体的摩尔比转化成One Shot TOP10感受态细胞。
    2. 生成每个样品20个克隆
    3. 按照制造商的说明书,使用GeneJet Plamsid Miniprep Kit纯化质粒。
    4. Eurofins进行E1/E2插入片段测序。
    5. 检查所有痕迹文件以排除存在双峰或不明确序列区域的序列。

  5. 数据处理和纠错
    1. 原始sff数据文件使用SFFFile工具进行管理。
    2.  低于预期扩增子长度的90%的低质量读取和读取被移除。
    3. 使用具有γ分布和不变位点(GTR + G + I)的一般时间 - 可逆模型(使用MEGA 6.0)(Tamura等,2013)克隆数据的系统发生分离。
    4.  具有引导值(1,000次重采样)的主分支> 85个被分类为(次)谱系(Palmer等人,2014年)。
    5. 随后将两个24-bp基序定义为每个(亚)谱系的HVR1氨基酸谱,并将其应用于序列分析管线。该基序的第一个15-bp跨越E1的保守的3'末端。剩余的9-bp包括在E2的5'末端HVR1的前3个氨基酸。
    6. 使用的图案的总数反映了主要HVR1随时间的观察到的变化。对于每个(亚)谱系,两个基序参考序列被认为是足够的
    7. 为了提高测序误差校正算法(KEC-ET)的灵敏度,根据相应图案的存在对UDPS数据进行分区。
    8. 为了确保分析数据的质量和不存在PCR和测序嵌合体,与该图案集中的最佳匹配序列具有超过3bp差异的读数被去除。
    9. KEC由三个阶段组成
      1. 在阶段1中,计算来自处理数据集的读取集合的k-mers(固定长度k的子串),并分析k-mers频率的分布。误差阈值被计算为分离两个不同分布的k-mers的最小频率。
      2. 在阶段2中,具有低于误差阈值的频率范围被认为是错误的并且用于识别和纠正误差。校正是基于不同因素的分析,包括连续错误k-mers段的长度,该段末端的核苷酸序列以及类似的正确k-mers的频率。纠错的过程反复迭代一次。
      3. 在第3阶段,包含在阶段2中未被校正的k-mers的读取被丢弃
    10. 使用KEC的以下参数:k = 25和i = 3

数据分析

在原始文章 http://jvi.asm.org/content/88/23/13709.short (Palmer等人,2014)。

笔记

将所有血清样品进行基因分型,并通过Molecular Virology Diagnostic&科克大学医院研究实验室,科克,爱尔兰。 https://www.ucc.ie/EN/meddept /人/利亚姆 - 范宁/mvdrl/

食谱

  1. 1x TAE
    4.84克三碱基
    1.15ml乙酸冰川
    2 ml 0.5 M EDTA 将dH 2 O添加到1 L

参考

  1. Palmer,BA,Dimitrova,Z.,Skums,P.,Crosbie,O.,Kenny-Walsh,E.and Fanning,LJ(2014)。  通过超临界焦磷酸测序法绘制的慢性丙型肝炎病毒复合体内病毒群体的进化和结构分析。 J Virol 88(23):13709-13721。
  2. Skums,P.,Dimitrova,Z.,Campo,DS,Vaughan,G.,Rossi,L.,Forbi,JC,Yokosawa,J.,Zelikovsky,A.and Khudyakov,Y。(2012)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22759430"target ="_ blank">对病毒扩增子的下一代测序进行有效的纠错。 BMC生物信息学 13 Suppl 10:S6。
  3. Tamura,K.,Stecher,G.,Peterson,D.,Filipski,A.and Kumar,S。(2013)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/24132122"target ="_ blank"> MEGA6:分子进化遗传分析版本6.0。Mol Biol Evol 30(12):2725-2729。 />
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Palmer, B. A., Dimitrova, Z., Skums, P., Crosbie, O., Kenny-Walsh, E. and Fanning, L. J. (2017). Ultradeep Pyrosequencing of Hepatitis C Virus to Define Evolutionary Phenotypes. Bio-protocol 7(10): e2284. DOI: 10.21769/BioProtoc.2284.
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