Leibniz Institute for Age Research-Fritz Lipmann Institute, Jena, Germany.
BMC Genomics. 2010 Apr 19;11:252. doi: 10.1186/1471-2164-11-252.
The beta-defensin gene cluster (DEFB) at chromosome 8p23.1 is one of the most copy number (CN) variable regions of the human genome. Whereas individual DEFB CNs have been suggested as independent genetic risk factors for several diseases (e.g. psoriasis and Crohn's disease), the role of multisite sequence variations (MSV) is less well understood and to date has only been reported for prostate cancer. Simultaneous assessment of MSVs and CNs can be achieved by PCR, cloning and Sanger sequencing, however, these methods are labour and cost intensive as well as prone to methodological bias introduced by bacterial cloning. Here, we demonstrate that amplicon sequencing of pooled individual PCR products by the 454 technology allows in-depth determination of MSV haplotypes and estimation of DEFB CNs in parallel.
Six PCR products spread over approximately 87 kb of DEFB and harbouring 24 known MSVs were amplified from 11 DNA samples, pooled and sequenced on a Roche 454 GS FLX sequencer. From approximately 142,000 reads, approximately 120,000 haplotype calls (HC) were inferred that identified 22 haplotypes ranging from 2 to 7 per amplicon. In addition to the 24 known MSVs, two additional sequence variations were detected. Minimal CNs were estimated from the ratio of HCs and compared to absolute CNs determined by alternative methods. Concordance in CNs was found for 7 samples, the CNs differed by one in 2 samples and the estimated minimal CN was half of the absolute in one sample. For 7 samples and 2 amplicons, the 454 haplotyping results were compared to those by cloning/Sanger sequencing. Intrinsic problems related to chimera formation during PCR and differences between haplotyping by 454 and cloning/Sanger sequencing are discussed.
Deep amplicon sequencing using the 454 technology yield thousands of HCs per amplicon for an affordable price and may represent an effective method for parallel haplotyping and CN estimation in small to medium-sized cohorts. The obtained haplotypes represent a valuable resource to facilitate further studies of the biomedical impact of highly CN variable loci such as the beta-defensin locus.
位于 8p23.1 染色体上的β-防御素基因簇（DEFB）是人类基因组中拷贝数（CN）变化最大的区域之一。虽然单个 DEFB 的 CN 已被提议作为几种疾病（如银屑病和克罗恩病）的独立遗传风险因素，但多部位序列变异（MSV）的作用尚不清楚，迄今为止仅在前列腺癌中报道过。通过 PCR、克隆和 Sanger 测序可以同时评估 MSV 和 CN，但这些方法劳动强度大、成本高，并且容易受到细菌克隆引入的方法学偏差的影响。在这里，我们证明通过 454 技术对个体 PCR 产物进行扩增子测序可以深入确定 MSV 单倍型并平行估计 DEFB 的 CN。
从 11 个 DNA 样本中扩增了大约 87kb 的 DEFB 六个 PCR 产物，包含 24 个已知的 MSV，并在 Roche 454 GS FLX 测序仪上进行了测序。从大约 142000 个读段中，推断出大约 120000 个单倍型（HC），每个扩增子的单倍型数量从 2 到 7 不等。除了 24 个已知的 MSV 之外，还检测到了另外两个序列变异。通过 HC 的比率估计了最小 CN，并与通过替代方法确定的绝对 CN 进行了比较。在 7 个样本中发现了 CN 的一致性，在 2 个样本中 CN 相差 1 个，在 1 个样本中估计的最小 CN 是绝对 CN 的一半。对于 7 个样本和 2 个扩增子，将 454 单倍型分析的结果与克隆/Sanger 测序的结果进行了比较。讨论了与 PCR 过程中嵌合体形成相关的内在问题以及 454 和克隆/Sanger 测序之间的单倍型分析差异。
使用 454 技术进行深度扩增子测序，每个扩增子可获得数千个 HC，价格实惠，可能是在中小规模队列中进行平行单倍型分析和 CN 估计的有效方法。获得的单倍型代表了一个有价值的资源，可促进对β-防御素基因座等高度 CN 可变基因座的生物医学影响的进一步研究。
