National Centre for Biological Science, Tata Institute of Fundamental Research, Bengaluru, Karnataka, 560065, India.
School of Life Science, The University of Trans-Disciplinary Health Sciences & Technology (TDU), Bengaluru, Karnataka, 560064, India.
BMC Genomics. 2023 Mar 23;24(1):141. doi: 10.1186/s12864-023-09233-4.
Illumina sequencing platform requires base diversity in the initial 11 cycles for efficient cluster identification and colour matrix estimation. This limitation yields low-quality data for amplicon libraries having homogeneous base composition. Spike-in of PhiX library ensures base diversity but reduces the overall number of sequencing reads for data analysis. To overcome such low diversity issues during amplicon sequencing on illumina platforms, we developed a high throughput single amplicon sequencing method by introducing 'N' (0-10) spacers in target gene amplification primers that are pooled for simple handling.
We evaluated the efficiency of 'N' (0-10) spacer-linked primers by targeting bacterial 16S V3-V4 region, demonstrating heterogeneous base library construction. The addition of 'N' (0-10) spacers causes sequencing frameshift at every base that leads to base diversity and produces heterogeneous high quality reads within a single amplicon library. We have written a python based command-line software,"MetReTrim", to trim the 'N' (0-10) spacers from the raw reads ( https://github.com/Mohak91/MetReTrim ). We further demonstrated the accuracy of this method by comparative mock community analysis with standard illumina V3-V4 primer method. The ZymoBIOMICS™ microbial community DNA standard was used as a control for this study. We performed data analysisusing the DADA2 pipeline where taxonomy was assigned using SILVA database as reference. We observed no difference between the communities represented by our method and standard illumina V3-V4 primer method.
This method eliminates the need for PhiX spike-in for single amplicon sequencing on illumina MiSeq platform. This allows for sequencing of more number of samples in a run and a reduction in the overall cost. Given that Illumina sequencing works on SBS chemistry irrespective of the platform (such as HiSeq, MiSeq, NextSeq, NovaSeq, etc.) we propose that this strategy of using 'N' (0-10) spacer-linked primer design can be adopted for generating high-quality single locus amplicon sequencing in a high throughput manner across the illumina platform subject to further validation.
Illumina 测序平台需要在最初的 11 个循环中具有碱基多样性，以有效地进行簇识别和颜色矩阵估计。对于碱基组成均一的扩增子文库，这种限制会导致低质量的数据。添加 PhiX 文库可确保碱基多样性，但会减少数据分析的总测序读数。为了克服在 Illumina 平台上进行扩增子测序时出现的低多样性问题，我们通过在目标基因扩增引物中引入“N”（0-10）间隔子来开发高通量单扩增子测序方法，这些间隔子便于混合处理。
我们通过靶向细菌 16S V3-V4 区域评估了“N”（0-10）间隔子连接引物的效率，展示了异质碱基文库的构建。在每个碱基处添加“N”（0-10）间隔子会导致测序框架移位，从而产生碱基多样性，并在单个扩增子文库中生成异质的高质量读数。我们已经编写了一个基于 Python 的命令行软件“MetReTrim”，用于从原始读取中修剪“N”（0-10）间隔子（https://github.com/Mohak91/MetReTrim）。我们通过与标准 Illumina V3-V4 引物方法的模拟群落分析比较，进一步证明了该方法的准确性。ZymoBIOMICS™微生物群落 DNA 标准品被用作本研究的对照。我们使用 DADA2 管道进行数据分析，其中使用 SILVA 数据库作为参考进行分类群分配。我们观察到，我们的方法和标准 Illumina V3-V4 引物方法所代表的群落之间没有差异。
该方法消除了在 Illumina MiSeq 平台上进行单扩增子测序时对 PhiX 加标 spike-in 的需求。这允许在一次运行中对更多数量的样本进行测序，并降低了总体成本。鉴于 Illumina 测序在 SBS 化学物质方面的工作原理与平台无关（如 HiSeq、MiSeq、NextSeq、NovaSeq 等），我们建议采用这种使用“N”（0-10）间隔子连接引物设计的策略，以在 Illumina 平台上以高通量方式生成高质量的单基因座扩增子测序，还需进一步验证。
