Project description:The utility of RADseq in an experimental setting is also demonstrated, based on our chasacterisation of an APOBEC mutation signature in an APOBEC3A transfected mouse cell line. 0D5 cells, derived from SSM3 cells, were co-transfected with a mixture containing pcDNA3.1 vectors expressing either APOBEC3A or APOBEC3B (kindly donated by Vincent Caval), pcDNA3.1 construct expressing deaminase null APOBEC3A linked to a uracil deglycosylase construct and a plasmid encoding mutant GFP and WT mCherry that is a reporter for APOBEC mutagenesis. Cells were grown, and gDNA extracted, prior to preparation of RADseq libraries using a PstI- MspI double-digest. Libraries underwent a Pippin Prep to select fragments in the size range of 220-520 bp (genomic sequence plus 148 bp of adapters). Single-end sequencing (1x101bp) was performed on an Illumina NovaSeq6000 utilizing v1.5 chemistry. Reads were aligned to mm10 using bwa mem and variants called using the GATK4 pipeline.
Project description:We carried out a cross species cattle-sheep array comparative genome hybridization (aCGH) experiment in order to identify copy number variations (CNVs) in the sheep genome analysing animals of Italian dairy breeds (Sarda, Bagnolese, Laticauda, Massese and Valle del Belice) using a tiling oligonucleotide array with ~385,000 probes designed on the bovine genome. We identified 135 CNV regions (CNVRs) covering about 10.5 Mb of the virtual sheep genome referred to the bovine genome (0.398%) with a mean and median equal to 77.6 kb and 55.9 kb, respectively. A comparative analysis between the identified sheep CNVRs and those reported in the cattle and goat genomes indicated that overlaps between sheep and goat and sheep and cattle CNVRs are highly significant (P<0.0001) suggesting that several chromosome regions might contain recurrent interspecies CNVRs. Many sheep CNVs affect genes with important biological functions. Further studies are needed to evaluate the functional relevance of these CNVs. In this study we made use of the high conservation and homology between the cattle and sheep genomes determined by their phylogenetic closeness to identify CNVs in sheep applying the same approach we carried out to identify CNVs in the goat genome. We used a custom tiling array including ~385,000 oligonucleotide probes designed on the Btau_4.0 version of the Bos taurus genome assembly and analysed genomic DNA samples of 11 sheep belonging to 6 different Italian dairy sheep breeds (2 Sarda, 2 Bagnolese, 2 Comisana, 2 Massese, 2 Laticauda and 1 Valle del Belice) compared to the reference DNA of another Sarda sheep.
Project description:Milk and dairy products are an essential food and an economic resource in many countries. Milk component synthesis and secretion by the mammary gland involve expression of a large number of genes whose nutritional regulation remains poorly defined. We aim at understanding the genomic influence on milk quality and synthesis by comparing two sheep breeds, with different milking attitude, Sarda and Gentile di Puglia, using sheep-specific microarray technology. From sheep ESTs deposited at NCBI, we generated the first annotated microarray developed for sheep with a covering of most of the genome. Whole tissue samples of mammary gland were collected from 4 lactating individuals of two sheep (Ovis aries) breeds, Gentile di Puglia and Sarda. Biopsies of lactating mammary tissue were taken at two lactation stages (first record, stage 01: 6 days after lambing; second record, stage 02: 44 days after lambing) in both breeds. Tissues from mammary gland were immersed in RNAlater (Sigma) immediately after biopsy and stored at -20°C.