Comparison of related genomes has emerged as a powerful lens for genome interpretation. Here, we report the sequencing and comparative analysis of 29 eutherian genomes. We confirm that at least 5.5% of the human genome has undergone purifying selection, and report constrained elements covering ~4.2% of the genome. We use evolutionary signatures and comparison with experimental datasets to suggest candidate functions for ~60% of constrained bases. These elements reveal a small number of new coding exons, candidate stop codon readthrough events, and over 10,000 regions of overlapping synonymous constraint within protein-coding exons. We find 220 candidate RNA structural families, and nearly a million elements overlapping potential promoter, enhancer and insulator regions. We report specific amino acid residues that have undergone positive selection, 280,000 non-coding elements exapted from mobile elements, and ~1,000 primate- and human-accelerated elements. Overlap with disease-associated variants suggests our findings will be relevant for studies of human biology and health.

Summary Gorillas are humans’ closest living relatives after chimpanzees, and are of comparable importance for the study of human origins and evolution. Here we present the assembly and analysis of a genome sequence for the western lowland gorilla, and compare the whole genomes of all extant great ape genera. We propose a synthesis of genetic and fossil evidence consistent with placing the human-chimpanzee and human-chimpanzee-gorilla speciation events at approximately 6 and 10 million years ago (Mya). In 30% of the genome, gorilla is closer to human or chimpanzee than the latter are to each other; this is rarer around coding genes, indicating pervasive selection throughout great ape evolution, and has functional consequences in gene expression. A comparison of protein coding genes reveals approximately 500 genes showing accelerated evolution on each of the gorilla, human and chimpanzee lineages, and evidence for parallel acceleration, particularly of genes involved in hearing. We also compare the western and eastern gorilla species, estimating an average sequence divergence time 1.75 million years ago, but with evidence for more recent genetic exchange and a population bottleneck in the eastern species. The use of the genome sequence in these and future analyses will promote a deeper understanding of great ape biology and evolution.

Background: Methods for assigning strains to bacterial species are cumbersome and no longer fit for purpose. The concatenated sequences of multiple house-keeping genes have been shown to be able to define and circumscribe bacterial species as sequence clusters. The advantage of this approach (multilocus sequence analysis; MLSA) is that, for any group of related species, a strain database can be produced and combined with software that allows query strains to be assigned to species via the internet. As an exemplar of this approach, we have studied a group of species, the viridans streptococci, which are very difficult to assign to species using standard taxonomic procedures, and have developed a website that allows species assignment via the internet.

Gray platelet syndrome (GPS) is a predominantly recessive platelet disorder characterized by a mild thrombocytopenia with large platelets and a paucity of α-granules; these abnormalities cause mostly moderate but in rare cases severe bleeding. We sequenced the exomes of four unrelated cases and identified as the causative gene NBEAL2, a gene with previously unknown function but a member of a gene family involved in granule development. Silencing of nbeal2 in zebrafish abrogated thrombocyte formation.

When detecting positive selection in proteins, the prevalence of errors resulting from misalignment and the ability of alignment filters to mitigate such errors are not well understood, but filters are commonly applied to try to avoid false positive results. Focusing on the sitewise detection of positive selection across a wide range of divergence levels and indel rates, we performed simulation experiments to quantify the false positives and false negatives introduced by alignment error and the ability of alignment filters to improve performance. We found that some aligners led to many false positives, whereas others resulted in very few. False negatives were a problem for all aligners, increasing with sequence divergence. Of the aligners tested, PRANK's codon-based alignments consistently performed the best and ClustalW performed the worst. Of the filters tested, GUIDANCE performed the best and Gblocks performed the worst. Although some filters showed good ability to reduce the error rates from ClustalW and MAFFT alignments, none were found to substantially improve the performance of PRANK alignments under most conditions. Our results revealed distinct trends in error rates and power levels for aligners and filters within a biologically plausible parameter space. With the best aligner, a low false positive rate was maintained even with extremely divergent indel-prone sequences. Controls using the true alignment and an optimal filtering method suggested that performance improvements could be gained by improving aligners or filters to reduce the prevalence of false negatives, especially at higher divergence levels and indel rates.

+ Correspondence should be addressed to CAA (c.albers@gen.umcn.nl), WHO (who1000@cam.ac.uk) or AC (as889@cam.ac.uk) . Author Contributions: AC performed zebrafish knock down, analysis of zebrafish gene sequence; LHW, collected clinical cases with anti-Vel, performed confirmatory Sanger sequencing and phenotyping by flow cytometry and haem-agglutination; JCS performed confirmatory Sanger sequencing and analyzed the genotyping data; MK and PB analyzed the RNA-Sequencing data; PAS performed SMIM1 transfection experiments, MF and SF performed isolation of precursor cells; BS, GJ, AT and NG performed the analysis of the evolutionary conservation of the SMIM genes; AAS performed genotyping; EA, erythroblast culture and transfection; EB performed zebrafish knock down experiment with input from DS; HS, HHWS, VGH, NV performed cell culture experiments and performed EMSA's and transfection experiments and Q-PCR for SMIM1; RSNF, JK, HJW and LF performed eQTL and gene ontology analysis; AG, MN, JP, JGS, HLJ, KR, MdH were responsible for identification of Vel-negative and Vel-weak individuals by typing >360,000 samples; HHDK performed RNA-Seq with supervisory input from HGS who leads and coordinates the BluePrint epigenome project; GK supervised exome-sequencing; AR analysed expression data from whole genome expression arrays and RNAseq; HS expression data and vectors; DS iron homeostasis and other relevant laboratory measurements; D.St. oversaw zebrafish experiments. NS provided pre-publication access to red blood cell GWAS meta-analysis; PH eQTL analysis, expression data, SMIM1 vectors, pre-publication access to red blood cell GWAS meta-analysis; EvdS and WHO designed the study, CAA performed exome sequence analysis, Sanger sequence analysis, genetic analysis and statistical analysis; AC, LHW, EvdS, WHO and CAA wrote the paper.

Table of Contents I. Peptide Synthesis and Purification SI-2 i. Table SI-1. Peptide Sequences SI-2 ii. Table SI-2. Peptide calculated and observed masses SI-3 II. Peptide Characterization SI-4 A. Circular Dichroism SI-4 i. Figure SI-1. Thermostability of aPP and variants SI-5 ii. Figure SI-2. Thermostability of YY2 and variants SI-6 iii. Table SI-3. Peptide Melting Temperatures SI-7 B. Flow Cytometry SI-7 C. Confocal microscopy SI-8 D. Cell Viability SI-8 i. Figure SI-3. Cell Viability SI-9 III. References Cited SI-9 Supplemental Information SI-2 I. Peptide Synthesis and PurificationTable SI-1. Sequences of peptides and miniature proteins described in this work. aPP GPSQPTYPGDDAPVEDLIRFYNDLQQYLNVVTRHRY 1 aPP 4R1 GPSQPTYPGDDAPVRDLIRFYRDLQRYLNVVTRHRY aPP 5R1 GPSQPTYPGDDAPVRDLIRFYRDLRRYLNVVTRHRY aPP 6R1 GPSQPTYPGDDAPVRDLRRFYRDLRRYLNVVTRHRY YY2 APPLPPRNRGEDASPEELSRYYASLRHYLNLVTRQRY 2 YY2 3R1 APPLPPRNRGEDASPEELSRYYRSLRHYLNLVTRQRY YY2 4R1 APPLPPRNRGEDASPRELSRYYRSLRHYLNLVTRQRY YY2 5R1 APPLPPRNRGEDASPRELRRYYRSLRHYLNLVTRQRY R 8 Y RRRRRRRRY Tat (48-60) GRKKRRQRRRPPQY 3 Penetratin RQIKIWFQNRRMKWKK 4 Transportan GWTLNSAGYLLKINLKALAALAKKIL 5 KLA KLALKLALKALKAALKLA 6Synthesis. All peptides were synthesized using standard solid-phase Fmoc chemistry on a 25 µmol scale with a Symphony ® multi-channel solid phase synthesizer (Protein Technologies, Inc., Tuscon, AZ). All α-amino acids and resins were purchased from Novabiochem (San Diego, CA) and solvents were purchased from American Bioanalytical (Natick, MA). All peptides were synthesized to carry free amines at their N-termini and carboxamides at their C-termini. Peptides were labeled on their N-termini on resin for at least one hour with fluorescein-5-EX, succinimidyl ester (Invitrogen, Carlsbad, CA, Cat. # F-6130), which was dissolved in 1 mL DMF and added to the reaction vessel with 20 µL N,N-diisopropylethylamine (EDIPA).Purification. Peptides were purified by reverse-phase HPLC using Grace Vydac C8 preparative or semi-preparative scale columns (300 silica, 10 µm particle size, 22 mm x 250 mm) and water/acetonitrile gradients containing 0.1% TFA. Peptide identity was confirmed by mass spectrometry on an Applied Biosystems Voyager-DE Pro MALDI-TOF mass spectrometer

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