454测序技术对HIV感染者干燥血斑中病毒进行抗药性检测

 

来自Genomeweb的消息，根据加拿大公共卫生局的研究人员,利用二代测序技术对血斑中艾滋病病毒基因的进行测序，低成本地检测HIV病毒抗药性。此方法比基于PCR的对于血浆样品进行的基因分型成本更低，还可以使更全面了解地艾滋病毒抗药性信息。

 

一项发表于Antiviral Therapy杂志的初步研究中，加拿大国家艾滋病病毒及反转录病毒实验室利用罗氏454 GS FLX系统对48位感染HIV-1病毒病人的血斑进行测序，研究病毒的抗药性。这些样本来自在2007年进行的一项研究，这些病人被诊断感染HIV-1，但尚未接受任何治疗。

 

这次研究主要是为了确定是否能够利用二代测序技术从已经干燥的血斑中，鉴定抗药性病毒株，并且降低其检测成本。实验中使用GS FLX系统进行测序，并将结果与Sanger方法进行比较。研究人员将样本进行序列标记并混合后在GS FLX上测序，获得3个相互重叠的扩增子序列，覆盖了整个HIV-1 PR基因以及RT基因的前237个编码子。

 

结果发现，454测序结果与Sanger方法相符，甚至检测到一个Sanger法没有检测到的突变。文章的作者说道：“混合样本的454焦磷酸测序是一个非常有效而经济的二代测序方法，尤其应用于干血斑的HIV病毒抗药性发生率鉴定。这一方法有助于在有限的资源及配置下，低成本高通量地获得HIV病毒抗药性数据。”

 

加拿大卫生局HIV遗传学实验室主任James Brooks认为将血斑用于检测相比血液有诸多优势，比如更容易进行收集储存。收集血液样本需要止血带，针头，符合标准诊所设置并从血液中分离细胞成分。而血斑只需要抽取很少的血液，在纸上进行干燥保存。除此之外，血液需要保存在零下八十度，这一条件对于许多实验室是一个挑战，而血斑可更简单而长期的保存。

 

同时，他表示这次基因分型的实验表明，对未接受治疗的艾滋病患者的血液和血斑的检测结果有很好的一致性，为下一步寻找低成本的病毒抗药性筛选方法奠定了基础。这一技术有望将原有实验基因分型价格和商业测试价格分别降低40%和90%。

 

Brooks说，“虽然454的测序技术相对于其他二代测序系统价格略高，但是454的长度长特点非常适合我们所关注的HIV基因组的区段。而Ion Torrent的技术也有一定的特长，但是目前它的读长还不够长。所以这次454成为了赢家。”

 

除了更加便宜之外，比起传统方法二代测序方法可以更灵敏的发现变异基因。在初步研究中，即使在出现频率大于20%的变异中，454发现了一个Sanger测序没有发现的变异（其频率约为20.4%）。如果测序深度进一步增加，二代测序可以发现频率在1%的变异。研究人员下一步将决定测序深度，主要因素在于低频率变异的临床意义及重要性。Brooks说，“要决定测序深度并不容易，因为有证据表明一些低频率的突变也导致抗药性，而低频率突变在人群中出现的几率很小。”测序的深度也是影响检测价格的因素之一。目前对于一些低频率突变引起部分患者抗药性的原因还不明确。

 

对于临床来说，另外一个关键步骤是如何自动化这项检测。从样本制备到生物信息流程的建立，实验小组正与加拿大微生物实验室合作开发相关软件。此外，对于已接受治疗的患者，采取血斑是否合理还需进一步论证，因为在接受治疗之后所采集的血斑中的病毒和血液中的病毒可能不同。

 

实验室的最终目标是建立一个从焦磷酸测序到直接产生抗药性报告的一体化检测方案。这一技术将推广到加拿大以外的其他国家。比如世界卫生组织HIV病毒抗药性实验室网络中的其他实验室。Brooks所在的实验室也接受来自其他国家的样本，通常是一些中低收入国家，实验室赞助所有的检测费用，一旦实验成本可以有效降低，那么就可以提高更多的服务。

 

除此之外，这一技术也有望推广到其他疾病比如肝炎。目前加拿大卫生局已经着手用该方法对于丙肝病人的血斑中的病毒进行分型，以监测丙型肝炎的发展趋势，这一技术无疑对于降低基因分型成本有极大帮助。

 

 

 

 

By Monica Heger

 

Next-generation sequencing of dried blood spots could be a more cost-effective method for monitoring HIV drug resistance than PCR-based genotyping from plasma samples and could also enable broader access to HIV drug resistance testing, according to researchers with the Public Health Agency of Canada.

 

In a pilot study, published recently in Antiviral Therapy, researchers from the National HIV and Retrovirology Laboratories in Canada used next-gen sequencing on Roche's 454 GS FLX to screen for drug resistance on previously collected dried blood spot samples from 48 patients with HIV-1.

 

The samples were part of a prior study, published in Antiviral Therapy in 2007, that compared dried blood spots to plasma for HIV genotyping in patients who were newly diagnosed with HIV-1 and had not yet received treatment.

 

In the current study, the researchers wanted to test the ability of next-generation sequencing to identify drug-resistance variants from the dried blood spot samples as a lower-cost method of monitoring drug resistance. They used the GS FLX for the sequencing, and compared the results to the genotyping results and to Sanger sequencing.

 

The researchers used a tagged, pooled, amplicon sequencing strategy on two lanes of the GS FLX, creating three overlapping amplicons from each patient encompassing the entire HIV-1 PR gene and the first 237 codons of the RT gene.

 

The team found that the results of the 454 sequencing were concordant with Sanger sequencing, and even picked up a variant that Sanger sequencing failed to detect.

"Pooled 454 pyrsosequencing is an efficient and cost-effective [next-gen sequencing] method for determining HIV drug resistance prevalence using [dried blood spot] specimens," the authors concluded. "This technique can lower barriers of cost and throughput and might improve accessibility to HIV [drug resistance] surveillance" in resource-limited settings.

 

Dried blood spots have a number of advantages over plasma, including ease of collection and storage, said James Brooks, chief of the National Laboratory for HIV Genetics at the Public Health Agency of Canada and senior author of the study. Collecting plasma requires tourniquets, needles, and sophisticated laboratory infrastructure to then separate out the plasma from the cellular components. Dried blood spots, on the other hand, require just a method for drawing a small amount of blood and the paper on which the blood is dried and stored.

 

Additionally, he added, plasma must be stored at negative 80 degrees C, which can be challenging in areas with unstable power supplies. Dried blood spots are much more durable.

 

The genotyping study published in 2007 established the "equivalence of dried blood spots and plasma collected in field conditions in drug-naïve people," Brooks said. The next step was to find a lower-cost method of screening those samples for drug resistance.

 

Current costs for genotyping HIV specimens from dried blood spots run between $125 per sample for in-house methods to more than $400 per sample for commercial kits, according to the researchers, who estimated that their method could reduce those costs by 40 percent compared to in-house methods and by nearly 90 percent compared to commercial testing.

 

While sequencing with the 454 machine is more expensive than other next-gen systems, Brooks said that the read lengths from the 454 are suited for the "particular region of the HIV genome that we're interested in looking at. He added that the Ion Torrent is an "interesting technology," but its read lengths are not yet long enough. "For our application, [454 is] the winner today."

 

The investigators found that aside from being cheaper than conventional genotyping methods, next-gen sequencing is also better at picking up rare variants.

 

In the pilot study, the researchers only looked at variants present in 20 percent of more of the reads, and even at that level Sanger sequencing still missed one variant present in 20.4 percent of reads.

 

Depending on the depth of coverage, however, next-gen sequencing can detect very rare variants, below 1 percent frequency.

 

Deciding how deep to sequence and thus what level of variants to screen for is one of the next steps the team must figure out. Brooks said that the team would likely reanalyze some existing studies to evaluate the clinical significance of variants at 10 percent frequency. "We can reliably pick those up in a cost-effective way," he said.

 

Drawing that cutoff point is tricky, said Brooks, because while there is some evidence that rare variants predict therapeutic failure, not everyone with rare variants fails.

 

"The field hasn't yet generated enough data for us to have a good understanding for why people fail [drug treatments] with a minor variant present at a certain frequency and other people do not," he said.

 

Another requirement before sequencing of dried blood spots could be implemented clinically is to automate as much of the sample prep and bioinformatics pipeline as possible, he said.

 

For this step, he said that the team is working with researchers from Canada's National Microbiology Laboratory in Winnipeg, which has a bioinformatics core that will help develop software for data analysis.

 

Additionally, he said, the team will test the technique on patients who have already received drug treatment. There has been some concern that collecting dried blood spots might not be a good sampling method for patients who have already received therapy because the virus could change after receiving treatment.

 

"The current circulating virus may be different from the virus that gets archived," Brooks said. "So we don't know whether it's going to be an appropriate way of collecting specimens in the treatment-experienced population."

 

Ultimately, Brooks said the goal is to create a pipeline that would "take the processed output from the pyrosequencing and generate a report that will have the drug resistance pattern."

 

The technique could then be implemented in Canada as well as other countries. For instance, the laboratory is part of the World Health Organization's HIV drug resistance laboratory network that is accredited for testing HIV samples for drug resistance.

"Ultimately, once we've established this pipeline, we'd like to use this technique as a way of conducting both domestic HIV drug resistance surveillance and to also use it for our work that we're involved in with [WHO]," said Brooks.

 

Brooks said that as part of that network, the lab receives requests from other countries — often low- or middle-income nations — to test samples. Typically the lab sponsors the costs of these genotyping tests, "but if we can do this in a more cost-effective manner, then we can deliver this service to a greater number of clients," he said.

 

The same technique could be applied to other diseases as well, such as hepatitis. Brooks said the he is currently conducting a surveillance of hepatitis C patients with the Public Health Agency of Canada, doing genotyping of dried blood spots. "There's no reason why we couldn't use the same [sequencing] technology to lower the costs of genotyping," he said.