Stop-gain deletion Marimastat start-loss stop-gainAll mutations were in the heterozygous state.enrichment and psychological well-being. These met or exceeded those set forth in the Guide for the Care and Use of Laboratory Animals from the National Research Council of the US National Academy of Sciences.ResultsAssembly of MacaMWe combined Sanger sequences from the reference rhesus macaque with newly generated Illumina whole genome and exome sequences from the same animal and created new de novo contig and scaffold assemblies using the MaSuRCA assembler [9]. Although there are many procedures for generating scaffolds, they all produce misassemblies with greater or lesser frequency [34-37]. To identify and correct misassembled scaffolds and assign scaffolds to chromosomes, we used independent mapping data and preliminary scaffold annotation. To properly assign genomic segments, we introduced 395 breaks in the assembled scaffolds. There were a total of 2312 scaffolds assigned to chromosomes. To guide the splitting of misassembled scaffolds and placement of scaffolds on chromosomes, we used BLASTn [10] to align human exons with rhesus scaffolds, identified rhesus radiation hybrid markers [11,12] within scaffolds, and used rhesus FISH maps to identify areas of synteny as well as species differences in chromosome structure [5,13,14]. We PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/12711626 term our new assembly: MacaM.Annotation of MacaM(from a target list of 19,063 genes, Additional file 1). To accomplish this, we used rhesus macaque Illumina mRNA-sequences to assemble a total of 11,712 transcripts representing 9,524 distinct genes (Additional file 3) using de novo (Table 2) and reference-guided transcriptome assemblers. We used these rhesus transcripts, as well as human data, in our new annotation of the rhesus genome. We were not able to annotate six genes in the assembly due to mutations in the reference animal (Table 3). We were able to annotate several MHC class II genes as well as major histocompatibility complex, class I, F (MAMU-F). However, the more polymorphic and repetitive MHC class I genes are difficult to assemble and annotate. Additional targeted sequencing of the reference animal, such as has been done for other rhesus macaques [38,39], is necessary to provide a high-quality assembly and annotation for these genes. Given the importance of rhesus macaques for immunological studies, finished sequencing of the reference rhesus MHC class I genes would be beneficial. The new rhesus PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/9221828 annotation (MacaM_Annotation_v7.6) is available as a GTF file (Additional file 3). Future updates to the assembly and annotation will be made available here: http://www.unmc.edu/rhesusgenechip/ index.htm#NewRhesusGenome.Comparison of MacaM with rheMac2 and CR_We annotated 16,050 genes and 18,757 transcripts with full-length coding sequences in the MacaM genomeTable 4 Chromosome assembly statisticsAssembly rheMac2 CR_1.0 MacaM # contigs 172351 399581 93402 Total bp of contigs 2646263223 2562947788The MacaM assembly has 2,721,371,100 bp of sequence placed on chromosomes with a N50 contig size of 64,032 bp, more than double the size of the original published assembly and five times the size of the CR_1.0 assembly (Table 4). To independently assess the completeness and accuracy of the three rhesus macaque assemblies, we aligned Ion Torrent reads from the reference rhesus macaque against each assembly. These reads had not been used in any of the assemblies, including MacaM. We were able to align 93, 94 and 98 of these rea.