Hgap v3

Total genomic DNA of K. gyiorum SWMUKG01 was extracted using Rapid Bacterial Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s protocol. The genomic DNA was sent to Sangon Biotech (Shanghai, China) for de novo whole genome sequencing. A combination of HiSeq 2500 Sequencer (Illumina, San Diego, CA, USA) and the PacBio RSII platforms (Pacific Biosciences, Menlo Park, CA, USA) was employed for whole genome sequencing. De novo genome assembly of filtered reads was conducted using the Hierarchical Genome Assembly Process workflow (HGAP, v3; Pacific Biosciences) [16 (link)].

DNA of isolate Izh-4 was submitted to WGS using SMRT sequencing on the Pacific BioScience Technology platform. The sequencing service was provided by the core facility located at the Norwegian Sequencing Centre (NSC) (www.sequencing.uio.no). DNA was extracted from 64 × 10⁹ cells using a Maxwell® 16 and a Maxwell LEV Blood DNA kit (Promega, Germany). The 20 kb library preparation protocol was employed. Size selection of the final library was performed using 0.4x Amp beads. The library was sequenced on a Pacific Biosciences RS II instrument using P6-C4 chemistry with 360 min movie time, two SMRT cells were used for sequencing due to poor loading. De novo assembly was performed using hierarchical genome assembly process (HGAP v3, Pacific Biosciences, SMRT Analysis Software v2.3.0) with default parameters (expected genome size 1.6 Mb, minimum target coverage 15X). RS_Resequencing.1 software (SMRT Analysis version v2.3.0) was used to map SMRT reads back to sequences in order to correct contigs after assembly clean-up. PacBio contigs were polished by mapping Illumina pair-end reads using Pilon v1.22.

Trimmed reads from Y strain were assembled using SPADES (v3.9.0)^{69 (link)}, with substrings within a sequence of (k) length (K-mers) for assembly of 21, 33, 55, 77, 99 and 127 K-mers. Bug2146 was assembled using HGAP v3 (Pacific Biosciences, SMRT Analysis Software v2.3.0)^{70 (link)}, seed sequence length and minimal coverage values were set to 6 kb and 15X, respectively.
Statistics from the assemblies were obtained and analyzed by linux command line program Biopieces tool kit⁷¹ .
The percentage of GC (%GC) was calculated in each entire contig and in a windowed mode (window = 1000 pb, step = 500 pb). The %GC distribution was obtained with public custom perl scripts⁷² .
Gene prediction and annotation was performed using Prodigal algorithm (v2.6.3)^{73 (link)} setting to predict just complete open reading frames (ORFs) by using standard translation eukaryotic table. Gene families were predicted by Markov cluster algorithm (MCL)^{74 (link)}, functional prediction was performed by Best reciprocal BLAST (Basic Local Alignment Search Tool) hit to all proteins available on Tritryp database for T. cruzi strains (e-value ≤1e-5). Annotations were inspected manually when possible using IGV browser^{75 (link)}. Single copy genes (SCG) and Monoglyceride lipase were identified and extracted from MCL and Blastp results.