Update on Rambouillet Assembly, the v5.0 Reference, and Plans for FAANG RNA Sequencing

High quality reference genomes are fundamental resources for biology. We report here a new sheep reference genome for the Rambouillet breed generated using the latest methods for producing and de novo assembling long reads. High quality annotation will be enabled by assays of the FAANG quality samples that have been collected from the reference animal.

A total of 200 Gb of sequence was generated from a single ewe, Benz2616, using the Pacific Biosciences (PacBio) technology. The data have a 12.6 kb N50 and 8.9 kb mean subread length. We used both Falcon and Celera Assembler to error correct and assemble the error corrected reads and selected the more complete product of the Celera Assembler for further processing. The preliminary assembly has a contig N50 of 2.2 Mb, a total length of 2.85 Gb, 365 contigs contain half of the genome sequence, and the longest contig is 16.3 Mb. The majority (89%) of 338,551 EST sequences align to the genome, with most (90%) having nearly complete alignments, aligning over more than 95% of their length. This version of the genome is more complete and has more contiguous alignment to these expressed sequences than the Texel breed Oar4.0 reference. Base quality of this assembly is high, with error rates less than 1% following assembly polishing using Arrow. The preliminary contigs have been scaffolded using Hi-C data and the Lachesis software, producing chromosome scale scaffolds. Further scaffold refinement is ongoing.

To provide the highest quality annotation of this high quality assembly, we have collected samples from over 100 tissues from the reference animal for additional FAANG assays. This was a large effort involving over 35 people in the sample collection, advance planning and coordination. In addition to the PacBio genome sequence and Hi-C data for the genome assembly, PacBio IsoSeq, miRNAseq, ATAC-Seq and other assays are planned.