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

Abstract

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.

Figure 1

Flowchart illustrating procedures for assembly and annotation of the MacaM rhesus macaque genome.

Figure 2

Correction of rheMac2 SHE gene misassembly in MacaM. A. rheMac2 genome. Exons 1, 2, 4, 5 and 6 of the Src homology 2 domain containing E. (SHE) gene are contained within scaffold NW_001108937.1. Exon 3 of this gene was assigned to scaffold NW_001218118.1. Scaffold NW_001108937.1 was correctly assigned to chromosome 1. However, scaffold NW_001218118.1 was mistakenly assigned to chromosome X. This resulted in an annotation of the rhesus SHE gene with missing sequence (corresponding to exon 3). Additional details on the misassembly of this gene in rheMac2 can be found in [3]. B. MacaM genome. All 6 exons of the SHE gene were found on scaffold 2317188291 of the MacaM assembly.

Figure 3

Alignment of rhesus macaque SHE proteins from different annotations with human protein. Human SHE protein accession: NP_001010846.1. MacaM: Protein derived from the MacaM rhesus macaque genome. rheMac2_N: Protein obtained from the NCBI annotation of rheMac2, accession. rheMac2_E: Protein obtained from the Ensembl annotation of rheMac2, accession ENSMMUT00000032345. CR_1.0: Protein obtained from the Chinese rhesus macaque genome produced by BGI [8]. Yellow highlighting indicates identical sequence in human and alternative rhesus macaque annotations with the exception of sequences that are only shared in rheMac2_E and CR_1.0 which are indicated by green highlighting. Exon boundaries are indicated by line separating amino acids.

Figure 4

mRNA expression validation. We sequenced RNA from 60 rhesus macaque PBMC samples of differing ranks using Illumina paired end sequencing. After filtering, we mapped reads to either the MacaM (green symbols) or rheMac2 (blue symbols) assemblies using the STAR algorithm; we used CUFFLINKS to assign transcripts and determine differentially expressed genes (DEGs). (A) Number of uniquely mapping reads in individual RNA samples mapped using the MacaM and rheMac2 assemblies. Individual samples mapped by either assembly are joined by lines. (B) Percentage of total filtered reads that uniquely mapped to each assembly. (C) Number of DEGs that were identified using CUFFDIFF2.1 for dominant animals at two time points using the MacaM and rheMac2 genomes.

Figure 5

Number of DEGs which were identified in an experiment analyzing social anxiety in rhesus macaques. CUFFDIFF2.1 was used to identify DEGs with two Ranks (R1 = dominant; R2 = subordinate) and three time points (T1 = baseline; T2 = T1 + 20 minutes; T3 = T1 + 260 minutes). Human intruder intervention occurred immediately before T2, after T1.