Stiller Mathias, Knapp Michael, Stenzel Udo, Hofreiter Michael, Meyer Matthias
Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.
Genome Res. 2009 Oct;19(10):1843-8. doi: 10.1101/gr.095760.109. Epub 2009 Jul 27.
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.
尽管高通量测序技术的出现使得对已灭绝生物进行全基因组测序成为可能，但在加速对高度降解的DNA进行靶向测序方面进展甚微。在此，我们提出了一种新颖且高度灵敏的方法，用于对古代和降解的DNA进行靶向测序，该方法将多重PCR直接与样本条形码标记和高通量测序相结合。使用这种方法，我们仅通过两次454生命科学公司（罗氏）的GS FLX测序运行，就从31个洞熊（ Ursus spelaeus ）样本中获得了一个96%完整的线粒体基因组数据集。与以往仅依赖短序列片段的研究不同，我们的数据重叠部分包含了近10 kb的重复线粒体基因组序列，从而能够明确区分洞熊的三个主要进化分支。我们的方法为从大量难以处理的样本（如博物馆标本、医学收藏品或法医样本）中同时生成数千碱基的重叠序列数据提供了机会。在我们的方法中，我们提出了一种构建条形码测序文库的新方案，该方案与所有当前的高通量技术兼容，并且可以完全在板设置中进行。
