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 | Complexa | 1 to 5 | One step PCR (34) | More | No | No | Not mentioned | Cluster passing filter = 94%, 89.5% bases > Q30 | Highc | Lundberg et al. 2013 [1] |
Improved dual-indexing approach with heterogeneity spacer | Complexa | 1 to 7 | One step (30) | More | ~ 8–16% (Avg) | Yes | Not mentioned | ~ 85–93% bases > Q30 (Avg) | Highc | Fadrosh et al. 2014 [12] |
Phasing amplicon sequencing (PAS) | Complexa | 1 to 7 | Two step (10, 20) | Less | 10–20% | Yes | Read one – 250–251 bases Read two – 180 bases | 93.3% bases > Q30 | Highc | Liyou Wu et al. 2015 [13] |
Triple-index amplicon sequencing | Complexa | 1 to 7 | Two Step (25/30/35, 5/10) | Less | 10% | Yes | Not Mentioned | Not mentioned | Highc | Muinck et al. 2017 [7] |
2-step PCR library preparation with heterogeneity spacer | Complexb | 1 to 7 | Two Step (20, 10) | Less | 20% | Yes | Miseq Reads Read one – 148 bases Read two – 31 bases | Not mentioned | Moderated | Holm et al. 2019 [3] |
Target region amplification with a series of 10 primers | Complexa | 1 to 7 | Two step (25, 8) | Less | 10% | No | Read one – 185 bases Read two-160 bases | Not Mentioned | Highc | Jensen et al. 2019 [9] |
Pool of ‘N’ (0–10) spacer-linked target specific primer | Simpleb | 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 | Lowe | This manuscript |