High-quality single amplicon sequencing method for illumina MiSeq platform using pool of ‘N’ (0–10) spacer-linked target specific primers without PhiX spike-in

Table 2 Comparison with previously published methods that addressed diversity issues with single amplicon sequencing in illumina platforms

Approach	Spacer/Tag Design	Frame Shift	PCR strategy	Chimera potential	PhiX Spike-In	Mock Evaluation	Position in reads where Q score drop < 30	Run Metrics	Experimental cost	Reference
Random tags and spacer	Complex^a	1 to 5	One step PCR (34)	More	No	No	Not mentioned	Cluster passing filter = 94%, 89.5% bases > Q30	High^c	Lundberg et al. 2013 [1]
Improved dual-indexing approach with heterogeneity spacer	Complex^a	1 to 7	One step (30)	More	~ 8–16% (Avg)	Yes	Not mentioned	~ 85–93% bases > Q30 (Avg)	High^c	Fadrosh et al. 2014 [12]
Phasing amplicon sequencing (PAS)	Complex^a	1 to 7	Two step (10, 20)	Less	10–20%	Yes	Read one – 250–251 bases Read two – 180 bases	93.3% bases > Q30	High^c	Liyou Wu et al. 2015 [13]
Triple-index amplicon sequencing	Complex^a	1 to 7	Two Step (25/30/35, 5/10)	Less	10%	Yes	Not Mentioned	Not mentioned	High^c	Muinck et al. 2017 [7]
2-step PCR library preparation with heterogeneity spacer	Complex^b	1 to 7	Two Step (20, 10)	Less	20%	Yes	Miseq Reads Read one – 148 bases Read two – 31 bases	Not mentioned	Moderate^d	Holm et al. 2019 [3]
Target region amplification with a series of 10 primers	Complex^a	1 to 7	Two step (25, 8)	Less	10%	No	Read one – 185 bases Read two-160 bases	Not Mentioned	High^c	Jensen et al. 2019 [9]
Pool of ‘N’ (0–10) spacer-linked target specific primer	Simple^b	1 to 10	Two step (25, 8)	Less	No	Yes	Read one – 265–270 bases Read two – 235–240 bases	Cluster passing filter = 97.5%, 95.3% bases > Q30	Low^e	This manuscript

^aComplex – Required careful planning to design of spacer/tag to maintain base balance at each position, increased workload in case of library preparation per sample, complex design of primer/tag/spacers/indices. (Wherever applicable)
^bSimple – Simple design of primer/spacer/tag making the adaptability of the method easy to use even by non-experts
^cHigh – Experimental setup tedious (requires 6–8 samples to be pooled for confirming base complexity) and PhiX spiking reduces the overall sequence read throughput and multiplexing options in a run
^dModerate – Experimental setup simple but requires PhiX spiking
^eLow – Experimental setup simple, faster, base complexing within individual library and no need for PhiX spiking allows to multiplexing of more number of samples in a run

ISSN: 1471-2164