Ram Daniela, Leshkowitz Dena, Gonzalez Dimitri, Forer Relly, Levy Itzchak, Chowers Michal, Lorber Margalit, Hindiyeh Musa, Mendelson Ella, Mor Orna
National HIV Reference Laboratory, Central Virology Laboratory, Ministry of Health, Tel-Hashomer, Ramat-Gan, Israel.
Bioinformatics Unit, The Nancy and Stephen Grand National Center for Personalized Medicine, Weizmann Institute, Rehovot, Israel.
J Virol Methods. 2015 Feb;212:12-6. doi: 10.1016/j.jviromet.2014.11.003. Epub 2014 Nov 11.
Population HIV-1 sequencing is currently the method of choice for the identification and follow-up of HIV-1 antiretroviral drug resistance. It has limited sensitivity and results in a consensus sequence showing the most prevalent nucleotide per position. Moreover concomitant sequencing and interpretation of the results for several samples together is laborious and time consuming. In this study, the practical use of GS Junior and MiSeq bench-top next generation sequencing (NGS) platforms as an alternative to Trugene Sanger-based population sequencing in the clinical HIV laboratory was assessed. DeepChek(®)-HIV TherapyEdge software was used for processing all the protease and reverse transcriptase sequences and for resistance interpretation. Plasma samples from nine HIV-1 carriers, representing the major HIV-1 subtypes in Israel, were compared. The total number of amino acid substitutions identified in the nine samples by GS Junior (232 substitutions) and MiSeq (243 substitutions) was similar and higher than Trugene (181 substitutions), emphasizing the advantage of deep sequencing on population sequencing. More than 80% of the identified substitutions were identical between the GS Junior and MiSeq platforms, most of which (184 of 199) at similar frequency. Low abundance substitutions accounted for 20.9% of the MiSeq and 21.9% of the GS Junior output, the majority of which were not detected by Trugene. More drug resistance mutations were identified by both the NGS platforms, primarily, but not only, at low abundance. In conclusion, in combination with DeepChek, both GS Junior and MiSeq were found to be more sensitive than Trugene and adequate for HIV-1 resistance analysis in the clinical HIV laboratory.
群体HIV-1测序是目前用于鉴定和跟踪HIV-1抗逆转录病毒药物耐药性的首选方法。其灵敏度有限，结果是一个显示每个位置最常见核苷酸的一致性序列。此外，同时对多个样本进行测序并解释结果既费力又耗时。在本研究中，评估了GS Junior和MiSeq台式下一代测序（NGS）平台在临床HIV实验室中作为基于Trugene桑格测序的群体测序替代方法的实际应用。使用DeepChek(®)-HIV TherapyEdge软件处理所有蛋白酶和逆转录酶序列并进行耐药性解读。比较了来自9名HIV-1携带者的血浆样本，这些样本代表了以色列的主要HIV-1亚型。通过GS Junior（232个替换）和MiSeq（243个替换）在9个样本中鉴定出的氨基酸替换总数相似且高于Trugene（181个替换），强调了深度测序相对于群体测序的优势。GS Junior和MiSeq平台之间超过80%的已鉴定替换是相同的，其中大多数（199个中的184个）频率相似。低丰度替换占MiSeq输出的20.9%和GS Junior输出的21.9%，其中大多数未被Trugene检测到。两个NGS平台都鉴定出更多的耐药性突变，主要但不限于低丰度突变。总之，与DeepChek结合使用时，发现GS Junior和MiSeq都比Trugene更灵敏，适用于临床HIV实验室中的HIV-1耐药性分析。
