SequencErr: measuring and suppressing sequencer errors in next-generation sequencing data.

BACKGROUND: There is currently no method to precisely measure the errors that occur in the sequencing instrument/sequencer, which is critical for next-generation sequencing applications aimed at discovering the genetic makeup of heterogeneous cellular populations. RESULTS: We propose a novel computational method, SequencErr, to address this challenge by measuring the base correspondence between overlapping regions in forward and reverse reads. An analysis of 3777 public datasets from 75 research institutions in 18 countries revealed the sequencer error rate to be ~ 10 per million (pm) and 1.4% of sequencers and 2.7% of flow cells have error rates > 100 pm. At the flow cell level, error rates are elevated in the bottom surfaces and > 90% of HiSeq and NovaSeq flow cells have at least one outlier error-prone tile. By sequencing a common DNA library on different sequencers, we demonstrate that sequencers with high error rates have reduced overall sequencing accuracy, and removal of outlier error-prone tiles improves sequencing accuracy. We demonstrate that SequencErr can reveal novel insights relative to the popular quality control method FastQC and achieve a 10-fold lower error rate than popular error correction methods including Lighter and Musket. CONCLUSIONS: Our study reveals novel insights into the nature of DNA sequencing errors incurred on DNA sequencers. Our method can be used to assess, calibrate, and monitor sequencer accuracy, and to computationally suppress sequencer errors in existing datasets.

Siglec-E Negatively Regulates the Activation of TLR4 by Controlling Its Endocytosis.

TLR4 signaling is critical for providing effective immune protection, but it must be tightly controlled to avoid inflammation-induced pathology. Previously, we reported extensive and direct interactions between TLR and Siglec families of pattern recognition receptors. In this study, we examined the biological significance of this interaction during infection. We show that Siglec-E is required for Escherichia coli-induced endocytosis of TLR4. Siglec-E-deficient dendritic cells infected with E. coli fail to internalize TLR4. This leads to sustained TLR4 on the cell surface and activation of NF-κB and MAPK p38, resulting in high levels of TNF-α and IL-6 compared with wild-type dendritic cells. In contrast to the signaling events occurring at the plasma membrane, as a result of the inability to internalize TLR4, Siglec-E-deficient dendritic cells were also defective for TRIF-mediated IFN-ß production in response to E. coli infection. Furthermore, we found that accumulation of ubiquitinated TLR4 and binding of E3 ubiquitin ligase Triad3A to TLR4 was increased significantly in bone marrow-derived dendritic cells from wild-type mice, but not from Siglec-E-deficient mice, after E. coli infection. This represents a newly discovered mechanism that regulates the signaling of TLR4 during E. coli infection.

Gene changes may minimize masculinizing and defeminizing influences of exposure to male cotwins in female callitrichine primates.

BACKGROUND: Sexual differentiation in female mammals can be altered by the proximity of male littermates in utero, a phenomenon known as the intrauterine position effect (IUP). Among simian primates, callitrichines (marmosets and tamarins) are likely candidates for IUP, since they exhibit obligate dizygotic twinning and fetuses share extensive vascularization in utero. In this paper, we determined whether female reproductive parameters are altered by gestating with a male twin and evaluated changes in genes associated with anti-Müllerian and steroid hormones in twinning callitrichine primates. METHODS: We assessed the impact of gestation with male cotwins on reproductive performance and survivorship in female marmosets (Callithrix) and lion tamarins (Leontopithecus), contrasting measures for females gestated with one or more littermates (M+) or no male littermates (0M). We compared targeted coding regions for genes involved in steroidal and anti-Müllerian hormone mediation of sexual differentiation for representatives of twinning callitrichines (Callithrix, Saguinus, and Leontopithecus) with closely related New World primates that produce single births (Saimiri and Callimico). RESULTS: IUP effects in females were absent in female callitrichine primates: age at first ovulation, average litter size, and the proportion of stillborn infants, and lifetime survivorship did not differ between M+ and 0M females. We documented multiple nonsynonymous substitutions in genes associated with steroid synthesis, transport, and cellular action (SRD5A2, CYP19A1, SHBG, and AR) and with anti-Müllerian hormone (AMH and AMHR2) in callitrichines. In the only callitrichine to produce single infants (Callimico), two genes contained nonsynonymous substitutions relative to twinning callitrichines (CYP19A1 and AMRHR2); these substitutions were identical with nontwinning Saimiri and humans, suggesting a reversion to an ancestral sequence. CONCLUSIONS: In spite of a shared placental vasculature with opposite-sex twins throughout embryonic and fetal development, female callitrichine primates gestated with a male cotwin exhibit no decrement in reproductive performance relative to females gestated with female cotwins. Hence, IUP effects on female reproduction in callitrichines are modest. We have identified mutations in candidate genes relevant for steroid hormone signaling and metabolism, and especially in AMH-related genes, that are likely to alter protein structure and function in the callitrichines. These mutations may confer protection for females from the masculinizing and defeminizing influences of gestating with a male cotwin.

Induction of Siglec-1 by Endotoxin Tolerance Suppresses the Innate Immune Response by Promoting TGF-ß1 Production.

Sepsis is one of the leading causes of death worldwide. Although the prevailing theory for the sepsis syndrome is a condition of uncontrolled inflammation in response to infection, sepsis is increasingly being recognized as an immunosuppressive state known as endotoxin tolerance. We found sialylation of cell surface was significantly increased on LPS-induced tolerant cells; knockdown of Neu1 in macrophage cell line RAW 264.7 cells resulted in enhanced LPS-induced tolerance, whereas overexpression of Neu1 or treatment with sialidase abrogated LPS-induced tolerance, as defined by measuring TNF-α levels in the culture supernatants. We also found that the expression of Siglec-1 (a member of sialic acid-binding Ig (I)-like lectin family members, the predominant sialic acid-binding proteins on cell surface) was specifically up-regulated in endotoxin tolerant cells and the induction of Siglec-1 suppresses the innate immune response by promoting TGF-ß1 production. The enhanced TGF-ß1 production by Siglec-1 was significantly attenuated by spleen tyrosine kinase (Syk) inhibitor. Knockdown of siglec-1 in RAW 264.7 cells resulted in inhibiting the production of TGF-ß1 by ubiquitin-dependent degradation of Syk. Mechanistically, Siglec-1 associates with adaptor protein DNAX-activation protein of 12 kDa (DAP12) and transduces a signal to Syk to control the production of TGF-ß1 in endotoxin tolerance. Thus, Siglec-1 plays an important role in the development of endotoxin tolerance and targeted manipulation of this process could lead to a new therapeutic opportunity for patients with sepsis.

Production of Transgenic Pigs with an Introduced Missense Mutation of the Bone Morphogenetic Protein Receptor Type IB Gene Related to Prolificacy.

In the last few decades, transgenic animal technology has witnessed an increasingly wide application in animal breeding. Reproductive traits are economically important to the pig industry. It has been shown that the bone morphogenetic protein receptor type IB (BMPR1B) A746G polymorphism is responsible for the fertility in sheep. However, this causal mutation exits exclusively in sheep and goat. In this study, we attempted to create transgenic pigs by introducing this mutation with the aim to improve reproductive traits in pigs. We successfully constructed a vector containing porcine BMPR1B coding sequence (CDS) with the mutant G allele of A746G mutation. In total, we obtained 24 cloned male piglets using handmade cloning (HMC) technique, and 12 individuals survived till maturation. A set of polymerase chain reactions indicated that 11 of 12 matured boars were transgene-positive individuals, and that the transgenic vector was most likely disrupted during cloning. Of 11 positive pigs, one (No. 11) lost a part of the terminator region but had the intact promoter and the CDS regions. cDNA sequencing showed that the introduced allele (746G) was expressed in multiple tissues of transgene-positive offspring of No.11. Western blot analysis revealed that BMPR1B protein expression in multiple tissues of transgene-positive F1 piglets was 0.5 to 2-fold higher than that in the transgene-negative siblings. The No. 11 boar showed normal litter size performance as normal pigs from the same breed. Transgene-positive F1 boars produced by No. 11 had higher semen volume, sperm concentration and total sperm per ejaculate than the negative siblings, although the differences did not reached statistical significance. Transgene-positive F1 sows had similar litter size performance to the negative siblings, and more data are needed to adequately assess the litter size performance. In conclusion, we obtained 24 cloned transgenic pigs with the modified porcine BMPR1B CDS using HMC. cDNA sequencing and western blot indicated that the exogenous BMPR1B CDS was successfully expressed in host pigs. The transgenic pigs showed normal litter size performance. However, no significant differences in litter size were found between transgene-positive and negative sows. Our study provides new insight into producing cloned transgenic livestock related to reproductive traits.

Genetic diversity in oxytocin ligands and receptors in New World monkeys.

Oxytocin (OXT) is an important neurohypophyseal hormone that influences wide spectrum of reproductive and social processes. Eutherian mammals possess a highly conserved sequence of OXT (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly). However, in this study, we sequenced the coding region for OXT in 22 species covering all New World monkeys (NWM) genera and clades, and characterize five OXT variants, including consensus mammalian Leu(8)-OXT, major variant Pro(8)-OXT, and three previously unreported variants: Ala(8)-OXT, Thr(8)-OXT, and Phe(2)-OXT. Pro(8)-OXT shows clear structural and physicochemical differences from Leu(8)-OXT. We report multiple predicted amino acid substitutions in the G protein-coupled OXT receptor (OXTR), especially in the critical N-terminus, which is crucial for OXT recognition and binding. Genera with same Pro(8)-OXT tend to cluster together on a phylogenetic tree based on OXTR sequence, and we demonstrate significant coevolution between OXT and OXTR. NWM species are characterized by high incidence of social monogamy, and we document an association between OXTR phylogeny and social monogamy. Our results demonstrate remarkable genetic diversity in the NWM OXT/OXTR system, which can provide a foundation for molecular, pharmacological, and behavioral studies of the role of OXT signaling in regulating complex social phenotypes.

Molecular variation in AVP and AVPR1a in New World monkeys (Primates, Platyrrhini): evolution and implications for social monogamy.

The neurohypophysial hormone arginine vasopressin (AVP) plays important roles in fluid regulation and vascular resistance. Differences in AVP receptor expression, particularly mediated through variation in the noncoding promoter region of the primary receptor for AVP (AVPR1a), may play a role in social phenotypes, particularly social monogamy, in rodents and humans. Among primates, social monogamy is rare, but is common among New World monkeys (NWM). AVP is a nonapeptide and generally conserved among eutherian mammals, although a recent paper demonstrated that some NWM species possess a novel form of the related neuropeptide hormone, oxytocin. We therefore characterized variation in the AVP and AVPR1a genes in 22 species representing every genus in the three major platyrrhine families (Cebidae, Atelidae and Pitheciidae). For AVP, a total of 16 synonymous substitutions were detected in 15 NWM species. No non-synonymous substitutions were noted, hence, AVP is conserved in NWM. By contrast, relative to the human AVPR1a, 66 predicted amino acids (AA) substitutions were identified in NWM. The AVPR1a N-terminus (ligand binding domain), third intracellular (G-protein binding domain), and C-terminus were variable among species. Complex evolution of AVPR1a is also apparent in NWM. A molecular phylogenetic tree inferred from AVPR1a coding sequences revealed some consensus taxonomic separation by families, but also a mixed group composed of genera from all three families. The overall dN/dS ratio of AVPR1a was 0.11, but signals of positive selection in distinct AVPR1a regions were observed, including the N-terminus, in which we identified six potential positive selection sites. AA substitutions at positions 241, 319, 399 and 409 occurred uniquely in marmosets and tamarins. Our results enhance the appreciation of genetic diversity in the mammalian AVP/AVPR1a system, and set the stage for molecular modeling of the neurohypophyseal hormones and social behavior in primates.

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.

Single nucleotide polymorphisms of caudal type homeobox 1 and 2 are associated with Barrett's esophagus.

BACKGROUND: Barrett's esophagus (BE), the premalignant lesion of esophageal adenocarcinoma, is believed to develop as a result of chronic gastroesophageal reflux disease (GERD). Approximately 10 % of subjects with GERD progress to BE. Genetic, epigenetic and other risk factors may contribute to this inter-individual variability. Caudal type homeobox 1 (Cdx1) and Caudal type homeobox 2 (Cdx2) play important regulatory roles in the development of human BE. AIMS: To determine associations between Cdx1 and Cdx2 single nucleotide polymorphisms (SNPs) and BE. METHODS: Genomic DNA was extracted from blood samples collected from BE (n = 109) and GERD (n = 223) patients for genotyping of 5 SNPs each of Cdx1 and Cdx2 using TaqMan allelic discrimination assays. Odds ratios and 95 % confidence intervals of SNPs and haplotypes were calculated with a logistic regression model adjusted for factors including age, sex and hiatal hernia. Interactions between genetic variants and these three risk factors were also analyzed. RESULTS: Older age (≥50 years), male sex and hiatal hernia were significantly associated with BE (P < 0.001). Five variants of Cdx1 SNPs (rs3776082, rs717746 and rs3776083), one Cdx1 haplotype, and three variants of Cdx2 SNPs (rs4769585 and rs3812863) were associated with BE (P < 0.05). Statistically significant interactions were detected between most of these SNPs and the three risk factors (P < 0.05). CONCLUSION: Certain SNPs of Cdx1 and Cdx2 and their interactions with other risk factors are associated with BE, and may contribute to human susceptibility to BE.

A whole genome scanning for quantitative trait loci on traits related to sperm quality and ejaculation in pigs.

To identify quantitative trait loci (QTL) for traits related to semen and ejaculation, phenotype data including semen volume, sperm concentration, total sperm per ejaculate, sperm motility, sperm abnormality rate, semen pH value, ejaculation times and ejaculation duration were measured on 206 F(2) boar at 240 days in a White Duroc x Erhualian intercross. A genome-wide scan was performed and the entire White Duroc x Erhualian intercross was genotyped for 183 microsatellite markers covering the whole pig genome. QTL analysis was performed using a composite regression interval mapping method via QTLExpress. A total of 18 QTL were detected, including 4 genome-wide significant QTL each for semen pH on pig chromosome (SSC) 2 and SSC12, for semen volume on SSC15, and for ejaculation times on SSC17. Fourteen suggestive QTL were found on SSC1, 2, 3, 4, 6, 9, 17 and 18. To our knowledge, this is the first report about the QTL for semen and ejaculation traits in pigs, providing a start point to decipher the genetic basis of these complex traits.

Genome-wide identification of QTL for age at puberty in gilts using a large intercross F2 population between White Duroc x Erhualian.

Puberty is a fundamental development process experienced by all reproductively competent adults, yet the specific factors regulating age at puberty remain elusive in pigs. In this study, we performed a genome scan to identify quantitative trait loci (QTL) affecting age at puberty in gilts using a White Duroc x Erhualian intercross. A total of 183 microsatellites covering 19 porcine chromosomes were genotyped in 454 F2 gilts and their parents and grandparents in the White Duroc x Erhualian intercross. A linear regression method was used to map QTL for age at puberty via QTLexpress. One 1% genome-wise significant QTL and one 0.1% genome-wise significant QTL were detected at 114 cM (centimorgan) on SSC1 and at 54 cM on SSC7, respectively. Moreover, two suggestive QTL were found on SSC8 and SSC17, respectively. This study confirmed the QTL for age at puberty previously identified on SSC1, 7 and 8, and reports for the first time a QTL for age at puberty in gilts on SSC17. Interestingly, the Chinese Erhualian alleles were not systematically favourable for younger age at puberty.

Genetic variation of porcine prostaglandin-endoperoxide synthase 2 (PTGS2) gene and its association with reproductive traits in an Erhualian x Duroc F2 population.

Identification of major genes that genetically impact female fertility is important for successful selection of high prolificacy pig lines. Because it is the rate-limiting enzyme in the conversion of arachidonic acid to prostaglandins (PGs), which are important for ovulation, fertilization, implantation, decidualization and parturition, prostaglandin-endoperoxide synthase 2 gene (PTGS2) is a potential candidate gene affecting porcine reproductive traits. In this study, a PCR-RFLP was used to genotype a total of 1 031 animals, including 661 from twelve Chinese local pig breeds, 190 from three Western pig breeds and 180 F2 sows from Nanchang pig resource family. Differences in frequency distributions of PTGS2 among twelve Chinese and three Western pig breeds and populations generally agree with their prolificacy. The allele frequencies in Lower Changjiang River Basin Type pig breeds, North China Type and Central China Type breeds are significantly different from those in South China Type, Plateau Type and Western pig breeds (P<0.001). And no significant differences were observed among Lower Changjiang River Basin Type, North China Type, Central China Type pig breeds, between South China Type and Western pig breeds, in part because of similar fertility patterns. And notable associations as well as reliable additive and dominant effects were not detected in an Erhualian x Duroc F2 population (P>0.05). Whereas, there is a trend for animals with one copy of the favourable A allele to have an increased TNB (total number of piglet born) and TBA (the number of piglets born alive) and a decreased SB (stillborn pigs) trait. Considering its crucial role in reproductive pathways, the PTGS2 gene deserves further study.