文章：Development of a Dual-Index Sequencing Strategy and CurationPipeline for Analyzing Amplicon Sequence Data on the MiSeqIllumina Sequencing Platform

   测序平台越来越丰富，考虑选择测序平台的时候主要考虑如下因素：1）We contend thatsequence quality is the most important consideration，2）A second important consideration is the number ofreads that one can obtain per run and per dollar，3）A third consideration for 16S rRNA studies is thelength of the sequences, as longer sequences are easier to assign toa taxonomic group using a classifier.

   对于Illumina测序平台来讲，测序reads质量影响因素主要是：1）sequence samples with low genetic diversity，2）the amount of DNA loaded ontothe flow cell, as this affects the cluster density and the ability of theimage analysis software to discriminate between clusters.

   常用的建库方式是两步PCR，但是两步PCR容易出现：increasing the risks of artifacts commonly observed with large numbers of PCR rounds. 为此，一步PCR的方式出现，引物会包括：the Illumina adapter sequence、an index sequence (onlyfor the reverse primer) 、a 10-nt pad to prevent hairpin formation、a 2-nt linker that is noncomplementary to the 16S rRNA gene和a gene-specific primer。文库的相关示例图如下：

   得到的序列质量情况如下：

   通过错误率来看数据的质量：1）reads1的质量相对reads2较好；2）reads末端出现错误的概率更高；3）substitution、insertion和deletions更容易出现在质量值较低的区域；

   通过研究overlap区域的碱基错误率和留存reads比例研究数据质量：