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Direct multiplex sequencing (DMPS)-a novel method for targeted high-throughput sequencing of ancient and highly degraded DNA

机译：直接多重测序（DMPS）-一种针对古代和高度降解的DNA的靶向高通量测序的新方法

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Although the emergence of high-throughput sequencing technologies has enabled whole-genome sequencing from extinct organisms, little progress has been made in accelerating targeted sequencing from highly degraded DNA. Here, we present a novel and highly sensitive method for targeted sequencing of ancient and degraded DNA, which couples multiplex PCR directly with sample barcoding and high-throughput sequencing. Using this approach, we obtained a 96% complete mitochondrial genome data set from 31 cave bear (Ursus spelaeus) samples using only two 454 Life Sciences (Roche) GS FLX runs. In contrast to previous studies relying only on short sequence fragments, the overlapping portion of our data comprises almost 10 kb of replicated mitochondrial genome sequence, allowing for the unambiguous differentiation of three major cave bear clades. Our method opens up the opportunity to simultaneously generate many kilobases of overlapping sequence data from large sets of difficult samples, such as museum specimens, medical collections, or forensic samples. Embedded in our approach, we present a new protocol for the construction of barcoded sequencing libraries, which is compatible with all current high-throughput technologies and can be performed entirely in plate setup.
机译：尽管高通量测序技术的出现使来自灭绝生物的全基因组测序成为可能，但在加速高度降解的DNA的靶向测序方面进展甚微。在这里，我们提出了一种针对古代和降解的DNA的靶向测序的新颖且高度灵敏的方法，该方法直接将多重PCR与样品条形码和高通量测序相结合。使用这种方法，我们仅使用两次454 Life Sciences（Roche）GS FLX运行就从31个熊熊（Ursus spelaeus）样本中获得了96％的完整线粒体基因组数据集。与仅依赖短序列片段的先前研究相反，我们数据的重叠部分包含近10 kb的复制线粒体基因组序列，从而可以清楚地区分三个主要的洞穴熊进化枝。我们的方法为从大量困难样品（例如博物馆标本，医疗收藏或法医样品）中同时生成千千个重叠序列数据提供了机会。嵌入到我们的方法中，我们提出了一种用于构建条形码测序文库的新协议，该协议与所有当前的高通量技术兼容，并且可以完全在板设置中执行。

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