To conduct the library preparation for metabarcoding of the apicomplexan 18S rRNA gene on the MinION Mk1B sequencer (Oxford Nanopore Technologies, Oxford, UK) we utilised the PCR Barcoding Expansion 1-96 (EXP-PBC096) with ONT’ Ligation Sequencing Kit (SQK-LSK110). The protocol followed was ‘Ligation sequencing amplicons - PCR barcoding (SQK-LSK110 with EXP-PBC096)’ version: PBAC96_9114_v110_revK_10Nov2020, with some modifications to improve yield.
For the first step PCR amplification 20 µl PCRs were conducted using 10 µl of LongAmp® Hot Start Taq 2× Master Mix (New England Biolabs, Massachusetts, USA) 7 µl Ambion nuclease-free water (Life Technologies, California, USA) from hereon referred to as water, 1 µl of forward primer APICO_F_Mod_ONT, 1 µl of reverse primer Api_Illum_New-Rev_ONT and 1 µl of genomic blood extracted DNA from Cambodian dogs. The original primer sequences were modified to include the addition of ONT adapter sequences (underlined) that permit the addition of DNA barcodes in a subsequent secondary PCR reaction, hence the primer sequences were APICO_F_Mod_ONT: 5’-TTTCTGTTGGTGCTGATATTGCGCCAGTAGTCATATGCTTGTCT-3’ and Api_Illum_New-Rev_ONT: 5’-ACTTGCCTGTCGCTCTATCTTCCTGTTATTGCCTYAAACTTCCTYG-3’.
PCRs were then conducted on a T100TM Thermal Cycler (Bio-Rad, California, USA) using the following conditions: 1 cycle of 94 °C for 1 min, 20 cycles of 94 °C for 30 s, 56 °C for 45 s and 65 °C for 1 min 20 s, with a final extension of 65 °C for 10 min. Separate and different physical laboratory areas were utilised for DNA extraction, pre-PCR and post-PCR experiments with all first-step PCRs prepared in a PCR hood under sterile conditions with filter tips, following UV sterilisation of the workspace.