title: NGS of giardia intestinalis gluatmate dehydrogenase (gdh) gene: human fecal isolate
design: Samples and DNA extraction:Giardia positive human samples were submitted anonymously from accredited national diagnostic laboratories for our routine surveillance work (Garcia-R et al., 2017). Two faecal samples received in our lab in January 2016 and October 2017 that were sent from diagnostic laboratories in Christchurch and Dunedin (South Island, New Zealand) were stored at 4 C until further analysis. DNA extraction was performed as indicated in Garcia-R et al. (2017). Briefly, DNA extraction was carried out using the Isolate fecal DNA (Bioline) kit with disruption of the cysts using a beadbeater (Tissue Lyser II, Qiagen) at 30 Hz for 5 min.Next-Generation Sequencing:Amplicon Next-Generation Sequencing (NGS) can help to determine the presence of low-abundance intra-isolate variants in mixed infections (Horton, Bridle, Alexander, & Katzer, 2019). Partial gdh gene sequences were amplied for NGS on the MiSeq (Illumina) platform using the same assay described for Sanger sequencing above, with the exception that secondary PCR primers were modied to contain MiSeq adapter sequences on the 5 end. Agarose gel electrophoresis was used to verify the presence of fragments of approximately 432 bp. The PCR products were cleaned according to Illumina recommended protocols and DNA concentration was measured using NanoDrop. Sequencing was performed on an Illumina MiSeq using 500-cycle V2 chemistry (250 paired-end reads) following the manufacturers recommendations. An Illumina prepared PhiX library was loaded onto the Illumina MiSeq run at 20% volume as control.