High-Throughput De Novo Sequencing of Laser Microdissected Y Chromosome in the Mediterranean River Buffalo (2n = 50,XY)

The sequencing and correct assembly of the Y chromosome sequences is still a challenge in mammals. In fact, apart from the pseudoautosomal regions (PARs), the Y chromosome lacks a homologous counterpart in the X chromosome. Therefore, in males, the use of genomic DNA as a template for NGS allows isolating the specific Y sequences only as a difference from the X sequences, with many potential gaps and assembly errors for the contemporary presence of X and Y. To overcome this problem, we present a highthroughput sequencing approach based on the direct isolation of Y-chromosomes by laser microdissection in the Mediterranean river buffalo. Peripheral blood lymphocytes from 10 buffalo bulls were cultured in vitro for normal cultures. Fixed lymphocytes were spread on a polyethylene naphthalate membrane (PEN), which was attached to a 24 × 60 glass slide and treated for GTG-banding. Ten copies of the Y-chromosome from each bull were laser microdissected and collected in individual PCR tubes for DOP-PCR amplification and labeling. FISH confirmed the specific hybridization of each Y-probe on lymphocyte metaphases before NGS. Library preparation was performed with the NEB Next Ultra II DNA Library Prep Kit for Illumina. High-throughput sequencing was performed by Illumina technology with the NovaSeq 6000 S4 Reagent Kit v1.5 (300 cycles). Raw data were processed by TrimGalore (v0.6.7), and de novo assembly was accomplished by SPAdes genome assembler v3.15.5. Gene sequences were predicted by AUGUSTUS (v3.5.0). We generated about 40 Gb (90×) of Illumina short reads. Total assembly length was 2,260,027 bp, with an average GC% of 48.11%. A total of 861 genes were predicted, and 807 of them have a hit-to-reference, 210 are uncharacterized, and around 30 are without description. The total number of microsatellites identified was 552. Variant calling was conducted using the GATK4 pipeline, specifically employing the HaplotypeCaller tool for each sample separately and also in a multi-sample version. The total number of variants across all samples was 25,100. Our approach yielded valuable insights into the genomic characteristics of the Y chromosome, and our results represent a milestone for the river buffalo.