The heterogeneity spacers are short sequences linked to index adaptors or to the gene specific amplification primers in the form of 0-7 bases and minimizes the need for PhiX spike-in to 10 % by introducing base complexity at the start of sequencing reads yielding high quality sequencing and increased multiplexing capacity ( Muinck et al., 2017 Holm et al., 2019 Herbold et al., 2015 Kozich et al., 2013). Another approach to deal with this issue is by sequencing libraries tagged with heterogeneity spacers at the 5’ end of the target gene amplicon during library preparation. Even though the research field has progressed in successful sequencing of 16S rRNA with Illumina V3–V4 region primers, the problems of drop in read quality and inherent error rate still remain unresolved ( Jensen et al., 2019). The base diversity in first few cycles, particularly in the first 11 bases of the amplicon, are crucial for identification of the sequencing clusters on the flow cell and color matrix estimation ( Jensen et al., 2019 Holm et al., 2019). However, it reduces the overall sequence read throughput and multiplexing options because of it being a non-target (PhiX) library ( Holm et al., 2019 Muinck et al., 2017). Libraries of low sequence diversity like 16s rRNA gene are highly homogenous and commonly spiked with high-diversity library such as PhiX, to alleviate the problem of homogenous signals generated across the entire flow cell. Therefore, for effective template generation and accurate base-calling on Illumina platforms it is required to have nucleotide diversity (equal proportions of A, C, G, and T nucleotides) at each base position in a sequencing library ( Muinck et al., 2017 illumina 2014). The similar emission spectra of the fluorophores (A and C as well as G and T) and limitations of the filters increases chances of low base call quality and mismatch rate in homogeneous sequence libraries ( Schirmer et al., 2015). Different filters are employed to image and identify the four different nucleotides. Red laser illuminates A and C and green laser illuminates G and T fluorophores. Illumina’s sequencing-by-synthesis technology uses fluorescently labelled reversible terminator-bound dNTPs. Illumina HiSeq and MiSeq sequencing platforms are extensively used for performing paired-end sequencing to generate millions of reads for amplified fragments of the 16S rRNA gene, the internal transcribed spacer (ITS) region and different marker genes ( Holm et al., 2019). This method terminates the need for PhiX spike-in and allows for multiplexing of multiple samples, greatly reducing the overall cost and yields improved sequence quality.Īmplicon sequencing is an important and widely used tool for inferring the presence of taxonomic groups in microbial communities, detecting genetic variation embedded in complex and genetic backgrounds, and is far more cost-effective than untargeted sequencing when large amounts of undesired genetic material is present ( Lundberg et al., 2013 Callahan et al., 2019). We have written a python script “MetReTrim” to trim the heterogenous ‘N’ spacers from the pre-processed reads. Addition of ‘N’ spacer causes sequencing frame shift at every base that leads to base diversity and produces heterogenous high quality reads within single amplicon library. We evaluated the efficiency of ‘N’ spacer primers by targeting bacterial 16S V3-V4 region, demonstrating heterogonous base library construction. To overcome such low diversity issues during amplicon sequencing on illumina platforms we developed high throughput single amplicon sequencing method by introducing ‘N’ spacers in target gene amplification primers that are pooled for simple handling. Spike-in of PhiX library ensures base diversity but overall reduces the number of sequencing reads for data analysis. This limitation yields low quality data for amplicon libraries having homogeneous base composition. Illumina sequencing platform requires base diversity in initial 11 cycles for efficient cluster identification and color matrix estimation.
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