NEW TECHNOLOGIES FOR TRYPANOSOMA CRUZI TYPING: A NEXT GENERATION SEQUENCING PIPELINE BASED ON HYPERVARIABLE REGIONS OF KDNA MINICIRCLES

RUSMAN, FANNY; TOMASINI, NICOLÁS; RAGONE PAULA GABRIELA; ROMANO MIRYAN; DIOSQUE, PATRICIO

Caxambú

Encuentro; XXXIV Annual Meeting of the Brazilian Society of Protozoology / XLV Annual Meeting on Basic Research in Chagas Disease; 2019

Sociedade Brasileira de Protozoologia

The kinetoplastic DNA of Trypanosoma cruzi consists of dozens of maxicicles and about 30,000 minicircles. Each minicircle (≈ 1.4 kb) is organized into four conserved regions (≈ 120 bp) and intercalated by an equal number of hypervariable regions (≈ 240 bp) known as mHVRs (minicircle hypervariable regions). There are few known mHVR sequences, possibly due to technical difficulties for sequencing this kind of DNA regions in the past. Nowadays, the Next Generation Sequencing (NGS) techniques open the possibility of a deeper knowledge of the mHVR sequence diversity. In the present work, we have optimized a mHVR targeted sequencing pipeline based on the Illumina MiSeq technology for the analysis of different strains of the main T. cruzi lineages (TcI-TcVI). Approximately 1.5 million reads were analyzed for each of the following strains: Tev55cl1 (TcI), PalDa20cl3 (TcI), Tu18 (TcII), Esmeraldo (TcII), X109 (TcIII), CANIII (TcIV), LL014R1 (TcV), MNcl2 (TcV) and LL015P68R0cl4 (TcVI). Sequences were clustered according to different identity thresholds (85%, 90% and 95%). In addition, different measures of alpha and beta diversity were calculated. Results show that sequencing at low depth (10.000 reads) is enough to put in evidence the main body of sequence diversity. It was also observed that strains of the same lineage clustered together in principal component analysis and UPGMA graphs. The number of clusters of sequences was variable among strains and some clusters were strain-specific or lineage-specific. The results of this work indicate that this reproducible NGS pipeline could be applied for genetic typing of T. cruzi. These are the first results in the framework of a broader work that aims to study the mHVR diversity and its evolution, as well as searching for lineage- specific sequences for typing.