Exome screening to identify loss-of-function mutations in the rhesus macaque for development of preclinical models of human disease.

BACKGROUND: Exome sequencing has been utilized to identify genetic variants associated with disease in humans. Identification of loss-of-function mutations with exome sequencing in rhesus macaques (Macaca mulatta) could lead to valuable animal models of genetic disease. Attempts have been made to identify variants in rhesus macaques by aligning exome data against the rheMac2 draft genome. However, such efforts have been impaired due to the incompleteness and annotation errors associated with rheMac2. We wished to determine whether aligning exome reads against our new, improved rhesus genome, MacaM, could be used to identify high impact, loss-of-function mutations in rhesus macaques that would be relevant to human disease. RESULTS: We compared alignments of exome reads from four rhesus macaques, the reference animal and three unrelated animals, against rheMac2 and MacaM. Substantially more reads aligned against MacaM than rheMac2. We followed the Broad Institute's Best Practice guidelines for variant discovery which utilizes the Genome Analysis Toolkit to identify high impact mutations. When rheMac2 was used as the reference genome, a large number of apparent false positives were identified. When MacaM was used as the reference genome, the number of false positives was greatly reduced. After examining the variant analyses conducted with MacaM as reference genome, we identified two putative loss-of-function mutations, in the heterozygous state, in genes related to human health. Sanger sequencing confirmed the presence of these mutations. We followed the transmission of one of these mutations (in the butyrylthiocholine gene) through three generations of rhesus macaques. Further, we demonstrated a functional decrease in butyrylthiocholinesterase activity similar to that observed in human heterozygotes with loss-of-function mutations in the same gene. CONCLUSIONS: The new MacaM genome can be effectively utilized to identify loss-of-function mutations in rhesus macaques without generating a high level of false positives. In some cases, heterozygotes may be immediately useful as models of human disease. For diseases where homozygous mutants are needed, directed breeding of loss-of-function heterozygous animals could be used to create rhesus macaque models of human genetic disease. The approach we describe here could be applied to other mammals, but only if their genomes have been improved beyond draft status.

A new rhesus macaque assembly and annotation for next-generation sequencing analyses.

BACKGROUND: The rhesus macaque (Macaca mulatta) is a key species for advancing biomedical research. Like all draft mammalian genomes, the draft rhesus assembly (rheMac2) has gaps, sequencing errors and misassemblies that have prevented automated annotation pipelines from functioning correctly. Another rhesus macaque assembly, CR_1.0, is also available but is substantially more fragmented than rheMac2 with smaller contigs and scaffolds. Annotations for these two assemblies are limited in completeness and accuracy. High quality assembly and annotation files are required for a wide range of studies including expression, genetic and evolutionary analyses. RESULTS: We report a new de novo assembly of the rhesus macaque genome (MacaM) that incorporates both the original Sanger sequences used to assemble rheMac2 and new Illumina sequences from the same animal. MacaM has a weighted average (N50) contig size of 64 kilobases, more than twice the size of the rheMac2 assembly and almost five times the size of the CR_1.0 assembly. The MacaM chromosome assembly incorporates information from previously unutilized mapping data and preliminary annotation of scaffolds. Independent assessment of the assemblies using Ion Torrent read alignments indicates that MacaM is more complete and accurate than rheMac2 and CR_1.0. We assembled messenger RNA sequences from several rhesus tissues into transcripts which allowed us to identify a total of 11,712 complete proteins representing 9,524 distinct genes. Using a combination of our assembled rhesus macaque transcripts and human transcripts, we annotated 18,757 transcripts and 16,050 genes with complete coding sequences in the MacaM assembly. Further, we demonstrate that the new annotations provide greatly improved accuracy as compared to the current annotations of rheMac2. Finally, we show that the MacaM genome provides an accurate resource for alignment of reads produced by RNA sequence expression studies. CONCLUSIONS: The MacaM assembly and annotation files provide a substantially more complete and accurate representation of the rhesus macaque genome than rheMac2 or CR_1.0 and will serve as an important resource for investigators conducting next-generation sequencing studies with nonhuman primates. REVIEWERS: This article was reviewed by Dr. Lutz Walter, Dr. Soojin Yi and Dr. Kateryna Makova.

De novo assembly of the common marmoset transcriptome from NextGen mRNA sequences.

BACKGROUND: Nonhuman primates are important for both biomedical studies and understanding human evolution. Although research in these areas has mostly focused on Old World primates, such as the rhesus macaque, the common marmoset (Callithrix jacchus), a New World primate, offers important advantages in comparison to other primates, such as an accelerated lifespan. To conduct Next Generation expression studies or to study primate evolution, a high quality annotation of the marmoset genome is required. The availability of marmoset transcriptome data from five tissues, including both raw sequences and assembled transcripts, will aid in the annotation of the newly released marmoset assembly. FINDINGS: RNA WAS EXTRACTED FROM FIVE TISSUES: skeletal muscle, bladder and hippocampus from a male common marmoset, and cerebral cortex and cerebellum from a female common marmoset. All five RNA samples were sequenced on the Illumina HiSeq 2000 platform. Sequences were deposited in the NCBI Sequence Read Archive. Transcripts were assembled, annotated and deposited in the NCBI Transcriptome Shotgun Assembly database. CONCLUSIONS: We have provided a high quality annotation of 51,163 transcripts with full-length coding sequence. This set represented a total of 10,833 unique genes. In addition to providing empirical support for the existence of these 10,833 genes, we also provide sequence information for 2,422 genes that were not previously identified in the Ensembl annotation of the marmoset genome.