Cycle sequencing optimization
Although the standard cycle sequencing protocol takes about 2.5 h, it has been shown that this time can be reduced to 50 min without compromising quality (Platt et al. 2007). We evaluated a series of cycle sequencing protocols (Table 2) to see if this time could be reduced further when dealing with a 300-bp amplicon. The shortest contiguous read lengths (CRLs) were obtained with protocols SEQ316–318, involving 10 s for denaturation and annealing stages (Table 2). Surprisingly, CRLs were longer in protocols with shorter denaturation and extension times, e.g. SEQ319–321 (5 s for denaturation and annealing) and SEQ315 vs. SEQ314 (15 s vs. 30 s denaturation). Overall, CRL was significantly decreased if the elongation time was shorter than 20 s. In the end, we were able to obtain satisfactory quality reads for ~300 bp in 26–31 min using programs SEQ319 and SEQ320.
Size-exclusion cleanup using the 4 K Nanosep device did not completely remove unincorporated dye-labelled terminators leaving ‘dye blobs’. Despite this fact, sequencing reactions cleaned up with Nanosep produced good quality sequences (CRL = 235–285) suitable for analysis using the BOLD identification engine.
DNA extracted from ethanol-fixed cotton swabs with alkaline lysis were used to evaluate the resistance of various enzymes to inhibitors. All fast enzymes, except KAPA 2G, were seriously inhibited by crude blood lysates (Fig. 3). Both KAPA 2G Fast (86%) and KAPA Ab (84%) outperformed Platinum Taq (80%), but KAPA Blood Direct (75%) and KAPA Robust kits (35%) often failed, apparently due to the formation of primer dimers. Phusion Flash High Fidelity kit (designed for whole blood amplification) performed inconsistently.
We also compared the performance of Z-Taq polymerase, KAPA 2G Fast, and KAPA Robust on FTA Elute cards to the performance of Platinum Taq on standard FTA cards extracted using the wildlife FTA protocol (Smith & Burgoyne 2004; Borisenko et al. 2008). Platinum Taq polymerase produced highest success on both FTA Elute disks and eluates (Fig. 4), while KAPA 2G Fast enzyme outperformed Z-Taq polymerase on FTA Elute disks.
Although overall PCR success with Platinum Taq was higher on standard FTA cards (100%), sequencing/identification success was higher for FTA Elute cards (88–90% vs. 78%). KAPA 2G Fast outperformed Z-Taq polymerase on FTA Elute disks, showing higher PCR (90% vs. 55%) and relatively high sequencing success (76%).
Protocols for DNA retrieval from FTA CloneSaver cards are tedious as they include multiple wash stages and special reagents. In contrast, the new FTA Elute technology requires only water for DNA retrieval. Moreover, FTA Elute disks can be used directly (without any wash) for PCR with new generation enzymes such as the KAPA Blood direct kit. In our experiments, the KAPA Blood Direct Kit showed 76% PCR success from unwashed FTA Elute disks. However, similar to results obtained with the crude alkaline lysates, we noticed an increased formation of primer dimers, possibly affecting PCR efficiency. We did not evaluate an improved version of the Blood Direct kit currently available from Kapa Biosystems.
We noticed that cotton swabs were occasionally picked up from their wells while peeling aluminium foil from the top of the plate, causing potential contamination, and we detected one likely cross-contamination event. As well, one sample on the FTA Elute card was contaminated with ascomycete fungi, which likely happened in the field. Finally, we encountered co-amplification of pseudogenes in the birds Oporornis formosus and Habia rubica. As a result, the sequences for these bird species were not uploaded to BOLD. Based on our previous experience, this problem with pseudogenes can be solved by amplification of a full-length barcode region.
In summary, KAPA 2G Fast enzyme showed the best performance among the enzymes tested in this study, both on alkaline lysates and blotting cards. FTA Elute cards were much more convenient to handle than FTA CloneSaver cards or ethanol-preserved blood on cotton swabs. Currently, Whatman is launching the production of FTA Elute cards in CloneSaver 96-well compatible format (Wingkei So, personal communication), which should make this technology compatible with the high-throughput analysis.
If extremely short cycling times (e.g. 10 min) and a compact footprint are required, then specialized thermocyclers, such as Piko Thermal Cycler (FINNZYMES) and thin-walled PCR strips or non-skirted plates, are the optimal choice for high performance PCR and sequencing protocols.