title: Mosquito3_ITS
design: Mosquito samples and high - throughput sequencing. We used the DNA from Ae. 117 aegypti , Ae. albopictus, and Cx. quinquefasciatus samples either collected from the field 118 or reared in the lab for high - throughput sequencing to examine the fungal microbiome 119 [34] . To characterize the fungal microbiome of these mosquito species, the internal 120 transcribed sequence (ITS) was sequenced. The region spanning ITS2 w as sequenced 121 according to the Illumina metagenomic sequencing protocol. Libraries were prepared 122 following the ITS Metagenomics Protocol which includes the use of indexes from the 123 Nextera XT Index Kit v2 (Illumina). Library preparation was done according to Illumina ITS 124 metagenomic protocol (Illumina) (Table S1, ITS primers) [38] . Libraries were sequenced 125 on the MiSeq System with the MiSeq Reagent Kit v3 (Illumina, Catalog No. MS - 102 - 3003). 126 All MiSeq runs were per formed with a run configuration of 2 x 151 cycles. To enable the 127 calculation of error - rate metrics and to increase nucleotide base diversity for more 128 accurate base - calling, all libraries were spiked with 5% PhiX Control v3 (Illumina, Catalog 129 No. FC - 110 - 300 1). The NCBI Genbank accession number for the raw sequencing data 130 reported here is XXXXXXXXX. 131 132 PNA blocker PCR with microbiome samples. To block host amplification, PNA 133 blocker was designed and synthesised (PNA Bio, USA). The PCR was performed with 1 134 M of each primer ( Table S1 ), 2 M PNA,1X KAPA master mix (NEB) and 50 ng of 135 template DNA. The PCR conditions were as follows : 3 min at 95  C for initial denaturation ; 136 30 cycles of 30 sec at 95  C , 30 sec at 70  C , 30 sec at 55  C , 30 sec at 72  C , 5 min at Commented [SH4]: Kamil please update this 8 137 72  C , then 30 sec at 70  C clamping step for PNA. The product was digested with Sph I 138 which cuts the fungal ITS amplicon but not the region in mosquitoes (Fig. S1) . The PCR 139 products were purified and sequenced as described above
library_name: Mosquito3_ITS