Sample preparation for Roche/454, ABI/SOLiD and Life Technologies/Ion Torrent sequencing are based on amplification of library fragments on the surface of beads prior to sequencing. Commonly, libraries are barcoded and pooled, to maximise the sequence output of each sequence run. Here, we describe a novel approach for normalization of multiplex next generation sequencing libraries after emulsion PCR. Briefly, amplified libraries carrying unique barcodes are prepared by fluorescent tagging of complementary sequences and then resolved by high-speed flow cytometric sorting of labeled emulsion PCR beads. The protocol is simple and provides an even sequence distribution of multiplex libraries when sequencing the flow-sorted beads. Moreover, since many empty and mixed emulsion PCR beads are removed, the approach gives rise to a substantial increase in sequence quality and mean read length, as compared to that obtained by standard enrichment protocols.
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机译：Roche / 454，ABI / SOLiD和Life Technologies / Ion Torrent测序的样品制备基于测序前珠表面的文库片段扩增。通常，对文库进行条形码处理和合并，以最大化每个序列运行的序列输出。在这里，我们描述了乳液PCR后用于多重下一代测序文库标准化的新方法。简而言之，通过对互补序列进行荧光标记来制备带有独特条形码的扩增文库，然后通过高速流式细胞术对标记的乳液PCR珠进行分类。该协议很简单，并且在对流分选的珠进行测序时，提供了多重文库的均匀序列分布。此外，由于去除了许多空的和混合的乳液PCR珠，与标准富集方案相比，该方法可显着提高序列质量和平均读取长度。
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