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Used default parameters for BLASTn alignments with the following exceptions: um_descriptions = 1; -num_alignments = 1; -max_ target_seqs = 1.Zimin et al. Biology Direct 2014, 9:20 http://www.biologydirect.com/content/9/1/Page 3 ofFigure 1 Flowchart illustrating procedures for assembly and annotation of the Lenvatinib MacaM rhesus macaque genome.1. We used BLASTn [10] to map exons from well-annotated human genes (Additional file 1) to scaffolds and re-ordered contigs so that, for protein coding orthologs, exons from each gene were in the correct order and orientation. This contiguity rule was used to enforce consistency whenever it was violated in subsequent steps. 2. There are several published reports of radiation hybrid mapping in rhesus macaques [11,12]. We used BLASTn to align markers identified in these studies with MaSuRCA scaffolds. We then used marker order information from the radiation hybrid studies to place scaffolds containing these markers in the correct order on chromosomes. 3. FISH mapping with human BACs has been used to identify syntenic blocks in rhesus macaques [5,13,14]. We cross-referenced these assignments with the locations of human genes within each block. We then used BLASTn exon ranges identified in step 1 to find the location of orthologous rhesus genes within the identified syntenic blocks. We placed scaffolds PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/12711626 containing these genes not already placed from step 2 on chromosomes according to the published synteny blocks. 4. There were still some scaffolds unplaced after step 3 as the radiation hybrid and FISH markers do not cover all portions of the rhesus chromosomes. To identify orthologous regions, we split human chromosome sequences into segments of 10,000 bp and used MegaBLAST [15] to align these segments against unplaced scaffolds. We then placed these scaffolds within the syntenic blocks defined by steps 2 and 3 in human chromosome order. As a result,small inversions and translocations may not be correctly represented. 5. Manual curation was used to resolve inconsistencies among the different sources of information. We developed a new chromosome nomenclature for the rhesus macaque (Table 1). Our goal was to designate chromosomes in accord with human and great ape nomenclature to facilitate comparison of rhesus macaque genes and chromosomes with these species. Chimpanzees, gorillas, and orangutans have the same general chromosomal structure as humans, with one notable exception. The human chromosome 2 appears to be the result of a fusion event that occurred during hominid evolution. Thus, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/22316373 both the great apes and rhesus macaques have two chromosomes that roughly correspond to the short and long arms of human chromosome 2. In the great apes, these two chromosomes are referred to as 2a and 2b. We adopted the same nomenclature for rhesus macaques to make comparisons between different primates easier. We have deposited our new rhesus macaque assembly (MacaM_Assembly_v7) in NCBI's BioProjects database under accession [GenBank:PRJNA214746].Chromosome assembly validationWe used genomic DNA from the reference rhesus macaque (animal 17573) to create a 400 bp library according to manufacturer's instructions (Ion Torrent, Personal Genome Machine). We sequenced this library on an Ion 318 chip and deposited 1.5 billion bases of sequence in the SRA under accession [GenBank:SRR1216390]. To independently assess genome assembly, we aligned theseZimin et al. Biology Direct 2014, 9:20 http://www.biologydirect.com/content/9/1/Page 4.