Sequencing of BAC pools by different next generation sequencing platforms and strategies.
Sequencing of BAC pools by different next generation sequencing platforms and strategies.
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DOI：
10.1186/1756-0500-4-411
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发表时间：
2011-10-14
影响因子：
1.8
通讯作者：
Platzer M
中科院分区：
文献类型：
--
作者：
Taudien S;Steuernagel B;Ariyadasa R;Schulte D;Schmutzer T;Groth M;Felder M;Petzold A;Scholz U;Mayer KF;Stein N;Platzer M
下一代BACs测序是一种可行的选择，甚至破译大而高度重复的基因组序列。为了优化这一策略，我们检测了读取长度对Roche/454序列组装质量的影响，Illumina/Solexa配对对（MPs）通过脚手架在多大程度上改善了组装，以及bac的条形码是否必要。使用FLX和Titanium技术对4个BACs进行测序显示出相似的测序精度，但表明较长的Titanium读取段产生的错误组装和间隙要少得多。96个条形码bac的454个片段与来自非条形码库的MPs进行了脚手架，改进了总组合体长度的79%。在没有条形码分离的情况下对454个序列进行组装，发现嵌合构象是一个主要问题，占总构象长度的47%。掩盖序列将这一比例降低到24%。最佳BAC池测序应基于最长的可用reads，而条形码对于重复和非重复序列信息的综合评估至关重要。当兴趣仅限于非重复区域，并且在组装之前重复被掩盖时，条形码是不必要的。在任何情况下，装配可以大大改进脚手架与非条形码的BAC池MPs。
Next generation sequencing of BACs is a viable option for deciphering the sequence of even large and highly repetitive genomes. In order to optimize this strategy, we examined the influence of read length on the quality of Roche/454 sequence assemblies, to what extent Illumina/Solexa mate pairs (MPs) improve the assemblies by scaffolding and whether barcoding of BACs is dispensable. Sequencing four BACs with both FLX and Titanium technologies revealed similar sequencing accuracy, but showed that the longer Titanium reads produce considerably less misassemblies and gaps. The 454 assemblies of 96 barcoded BACs were improved by scaffolding 79% of the total contig length with MPs from a non-barcoded library. Assembly of the unmasked 454 sequences without separation by barcodes revealed chimeric contig formation to be a major problem, encompassing 47% of the total contig length. Masking the sequences reduced this fraction to 24%. Optimal BAC pool sequencing should be based on the longest available reads, with barcoding essential for a comprehensive assessment of both repetitive and non-repetitive sequence information. When interest is restricted to non-repetitive regions and repeats are masked prior to assembly, barcoding is non-essential. In any case, the assemblies can be improved considerably by scaffolding with non-barcoded BAC pool MPs.