A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing.
A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing.
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DOI：
10.1093/nar/gkm760
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发表时间：
2007
影响因子：
14.9
通讯作者：
Fire AZ
中科院分区：
文献类型：
--
作者：
Parameswaran P;Jalili R;Tao L;Shokralla S;Gharizadeh B;Ronaghi M;Fire AZ
多路复用的高通量焦磷酸测序目前的复杂性有限（平行测序的样品数量）和容量（每个样品获得的序列数）。掩盖了可用的测序空间，以克服这些局限性，我们设计了一种新型的条形码方法促进了测序能力的后续分离。为每个定义的克隆链接组中的每组“ N'向前和“ N”反向条形码的序列，都可以从多达N2独立的库中进行序列。在使用GS20测序仪（454/Roche）的条形码测序中，我们发现，基于完美的前向或完美的反向条形码，可以将超过99.8％的获得的序列分配给25个独立的，独特的条形码库。据估计，发现速率通过序列的百分比为序列的百分比估计<0.005％。
Multiplexed high-throughput pyrosequencing is currently limited in complexity (number of samples sequenced in parallel), and in capacity (number of sequences obtained per sample). Physical-space segregation of the sequencing platform into a fixed number of channels allows limited multiplexing, but obscures available sequencing space. To overcome these limitations, we have devised a novel barcoding approach to allow for pooling and sequencing of DNA from independent samples, and to facilitate subsequent segregation of sequencing capacity. Forty-eight forward–reverse barcode pairs are described: each forward and each reverse barcode unique with respect to at least 4 nt positions. With improved read lengths of pyrosequencers, combinations of forward and reverse barcodes may be used to sequence from as many as n2 independent libraries for each set of ‘n’ forward and ‘n’ reverse barcodes, for each defined set of cloning-linkers. In two pilot series of barcoded sequencing using the GS20 Sequencer (454/Roche), we found that over 99.8% of obtained sequences could be assigned to 25 independent, uniquely barcoded libraries based on the presence of either a perfect forward or a perfect reverse barcode. The false-discovery rate, as measured by the percentage of sequences with unexpected perfect pairings of unmatched forward and reverse barcodes, was estimated to be <0.005%.
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影响因子：
3.1
作者：
Hofreuter, Dirk;Tsai, Jennifer;Galan, Jorge E.
通讯作者：
Galan, Jorge E.
影响因子：
14.9
作者：
Hoffmann C;Minkah N;Leipzig J;Wang G;Arens MQ;Tebas P;Bushman FD
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Bushman FD
影响因子：
82.9
作者：
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通讯作者：
Meyerson, Matthew
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作者：
Margulies, M;Egholm, M;Rothberg, JM
通讯作者：
Rothberg, JM
影响因子：
64.5
作者：
Ruby, J. Graham;Jan, Calvin;Bartel, David P.
通讯作者：
Bartel, David P.