title: Whole genome sequencing of Naumovozyma castelli: RNAi mutants
design: Genomic DNA was isolated using as described (Hoffman and Winston, 1987) from saturated YPD cultures of Naumovozyma castellii the genetic-selection strain and RNAi-mutant strains.  DNA concentration was calculated using the Qubit dsDNA HS (High Sensitivity) Assay Kit (Invitrogen Q32854) with an Invitrogen Qubit Fluorometer.  Genomic DNA libraries were prepared with either the Nextera DNA Library Preparation Kit (Illumina) or the QIAseq FX DNA Library Kit (QIAGEN 180475) and sequenced using Illumina SBS.  Single-end sequencing was performed on the HiSeq platform, and paired-end sequencing was performed on the HiSeq or NextSeq platforms. The N. castellii genome was downloaded from Yeast Genome Order Browser (YGOB - http://ygob.ucd.ie/), which is the same as the genome assembly ASM23734v1.  Sequences of the integrating plasmids containing GFP, URA3 and HIS3-PEST along with their silencing constructs, as well as the integrating plasmids containing extra copies of AGO1 and DCR1 were inserted into the FASTA file.  STAR (v.2.4) was used to create an indexed genome (Dobin et al., 2013).  Reads were aligned to the genome using STAR (v.2.4) with the parameters --outFilterMismatchNmax 4 --clip3pAdapterSeq CTGTCTCTTATAC --readMatesLengthsIn NotEqual --alignIntronMax 1 --alignMatesGapMax 1000. For samples that single and paired-end sequencing was performed, the BAM files were then concatenated before performing further analysis.