Illumina Miseq of hamoparasites from avian blood samples
The PCR1 mix was a 25 l reaction with 10 l QIAGEN Multiplex PCR Master Mix (Qiagen, Hilden, Germany), 2.5 l of each forward and reverse primer (3 M) tailed with Illumina sequencing primer sites (F: 5-TCTACACGTTCAGAGTTCTACAGTCCGACGATC-3 and R: 5GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3) and 1 l of DNA. The PCR thermal cycling programs were identical to those previously described for the amplified gene regions (see Table 1). Amplicons for each sample were normalised according to the intensity of the PCR product on a 1% agarose gel stained with ethidium bromide. PCR2 added sample-specific indexes: the 10 l PCR2 mix had 5 l QIAGEN Multiplex PCR Master Mix (Qiagen, Hilden, Germany), 1 l of each Illumina Fi5 and Ri7 indexes (1 M) whose combination was specific to each well (Integrated DNA Technologies, Belgium) and 4 l of the pooled amplicons for each sample from PCR1. The thermal cycler conditions were as follows: 15 minutes at 95C, then 10 cycles of 10 seconds at 98C, 30 seconds at 65C and 30 seconds at 72C, finishing with 5 minutes at 72C. Samples were normalised after quantifying on a FLUOstar OPTIMA (BMG Labtech, Ortenberg, Germany) using the QuantiFluor dsDNA system (Promega, UK) following the manufacturers instructions. The Agencourt AMPure XP system (Beckman Coulter, UK) was used for purification according to the manufacturers instructions. The purified products were eluted in 15 l of nuclease- free TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) with 1.5 l of 10 mM Tris-HCl/ 0.05% Tween 20 (pH 8.0) added. The prepared libraries were checked on the Agilent 4200 Tapestation (Agilent Technologies, CA, USA) for the expected peak amplicon size.Quantification of each pooled product was performed using qPCR. Triplicate dilutions of 1:100, 1:1000 and 1:10000 of the libraries were produced by serial dilution. The reagent mix and thermal cycling conditions were performed as per the manufacturers instructions (KAPA library quantification kit, KAPA Biosystems, UK). A StepOnePlus Real-Time PCR system (Applied Biosystems, CA, USA) was used to run the qPCR. The concentration of each library was calculated using the KAPA data analysis template and normalised to 4 nM. The library was sequenced using 250 paired-end reads on a MiSeq desktop sequencer (Illumina, San Diego, CA). Illumina samples were sequenced over four different Miseq runs, in combination with other samples, as part of a larger project (Thomas, 2017). Each run contained at least 10% duplicates (i.e 10 duplicates per 96 well plate).