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Public health newborn screening (NBS) programs provide population-scale ascertainment of rare, treatable conditions that require urgent intervention. Tandem mass spectrometry (MS/MS) is currently used to screen newborns for a panel of rare inborn errors of metabolism (IEMs) 1 – 4 . The NBSeq project evaluated whole-exome sequencing (WES) as an innovative methodology for NBS. We obtained archived residual dried blood spots and data for nearly all IEM cases from the 4.5 million infants born in California between mid-2005 and 2013 and from some infants who screened positive by MS/MS, but were unaffected upon follow-up testing. WES had an overall sensitivity of 88% and specificity of 98.4%, compared to 99.0% and 99.8%, respectively for MS/MS, although effectiveness varied among individual IEMs. Thus, WES alone was insufficiently sensitive or specific to be a primary screen for most NBS IEMs. However, as a secondary test for infants with abnormal MS/MS screens, WES could reduce false-positive results, facilitate timely case resolution and in some instances even suggest more appropriate or specific diagnosis than that initially obtained. This study represents the largest, to date, sequencing effort of an entire population of IEM-affected cases, allowing unbiased assessment of current capabilities of WES as a tool for population screening. Whole-exome sequencing is not sensitive or specific enough to replace the gold standard of tandem mass spectrometry screening of rare inborn errors of metabolism, but can help to reduce false positives and facilitate the timely resolution of ambiguous cases.

DIVERGE is a software system for phylogeny-based analyses of protein family evolution and functional divergence. It provides a suite of statistical tools for selection and prioritization of the amino acid sites that are responsible for the functional divergence of a gene family. The synergistic efforts of DIVERGE and other methods have convincingly demonstrated that the pattern of rate change at a particular amino acid site may contain insightful information about the underlying functional divergence following gene duplication. These predicted sites may be used as candidates for further experiments. We are now releasing an updated version of DIVERGE with the following improvements: 1) a feasible approach to examining functional divergence in nearly complete sequences by including deletions and insertions (indels); 2) the calculation of the false discovery rate of functionally diverging sites; 3) estimation of the effective number of functional divergence-related sites that is reliable and insensitive to cutoffs; 4) a statistical test for asymmetric functional divergence; and 5) a new method to infer functional divergence specific to a given duplicate cluster. In addition, we have made efforts to improve software design and produce a well-written software manual for the general user.

Background Curated databases of genetic variants assist clinicians and researchers in interpreting genetic variation. Yet, these databases contain some misclassified variants. It is unclear whether variant misclassification is abating as these databases rapidly grow and implement new guidelines. Methods Using archives of ClinVar and HGMD, we investigated how variant misclassification has changed over 6 years, across different ancestry groups. We considered inborn errors of metabolism (IEMs) screened in newborns as a model system because these disorders are often highly penetrant with neonatal phenotypes. We used samples from the 1000 Genomes Project (1KGP) to identify individuals with genotypes that were classified by the databases as pathogenic. Due to the rarity of IEMs, nearly all such classified pathogenic genotypes indicate likely variant misclassification in ClinVar or HGMD. Results While the false-positive rates of both ClinVar and HGMD have improved over time, HGMD variants currently imply two orders of magnitude more affected individuals in 1KGP than ClinVar variants. We observed that African ancestry individuals have a significantly increased chance of being incorrectly indicated to be affected by a screened IEM when HGMD variants are used. However, this bias affecting genomes of African ancestry was no longer significant once common variants were removed in accordance with recent variant classification guidelines. We discovered that ClinVar variants classified as Pathogenic or Likely Pathogenic are reclassified sixfold more often than DM or DM? variants in HGMD, which has likely resulted in ClinVar’s lower false-positive rate. Conclusions Considering misclassified variants that have since been reclassified reveals our increasing understanding of rare genetic variation. We found that variant classification guidelines and allele frequency databases comprising genetically diverse samples are important factors in reclassification. We also discovered that ClinVar variants common in European and South Asian individuals were more likely to be reclassified to a lower confidence category, perhaps due to an increased chance of these variants being classified by multiple submitters. We discuss features for variant classification databases that would support their continued improvement.

Background Window of implantation (WOI) displacement is one of the endometrial origins of embryo implantation failure, especially repeated implantation failure (RIF). An accurate prediction tool for endometrial receptivity (ER) is extraordinarily needed to precisely guide successful embryo implantation. We aimed to establish an RNA-Seq-based endometrial receptivity test (rsERT) tool using transcriptomic biomarkers and to evaluate the benefit of personalized embryo transfer (pET) guided by this tool in patients with RIF. Methods This was a two-phase strategy comprising tool establishment with retrospective data and benefit evaluation with a prospective, nonrandomized controlled trial. In the first phase, rsERT was established by sequencing and analyzing the RNA of endometrial tissues from 50 IVF patients with normal WOI timing. In the second phase, 142 patients with RIF were recruited and grouped by patient self-selection (experimental group, n = 56; control group, n = 86). pET guided by rsERT was performed in the experimental group and conventional ET in the control group. Results The rsERT, comprising 175 biomarker genes, showed an average accuracy of 98.4% by using tenfold cross-validation. The intrauterine pregnancy rate (IPR) of the experimental group (50.0%) was significantly improved compared to that (23.7%) of the control group (RR, 2.107; 95% CI 1.159 to 3.830; P  = 0.017) when transferring day-3 embryos. Although not significantly different, the IPR of the experimental group (63.6%) was still 20 percentage points higher than that (40.7%) of the control group (RR, 1.562; 95% CI 0.898 to 2.718; P  = 0.111) when transferring blastocysts. Conclusions The rsERT was developed to accurately predict the WOI period and significantly improve the pregnancy outcomes of patients with RIF, indicating the clinical potential of rsERT-guided pET. Trial registration Chinese Clinical Trial Registry: ChiCTR-DDD-17013375. Registered 14 November 2017, http://www.chictr.org.cn/index.aspx

RNA secondary structure may influence many cellular processes, including RNA processing, stability, localization, and translation. Single-nucleotide variations (SNVs) that alter RNA secondary structure, referred to as riboSNitches, are potentially causative of human diseases, especially in untranslated regions (UTRs) and noncoding RNAs (ncRNAs). The functions of somatic mutations that act as riboSNitches in cancer development remain poorly understood. In this study, we developed a computational pipeline called SNIPER (riboSNitch-enriched or depleted elements in cancer genomes), which employs MeanDiff and EucDiff to detect riboSNitches and then identifies riboSNitch-enriched or riboSNitch-depleted non-coding elements across tumors. SNIPER is available at github: https://github.com/suzhixi/SNIPER/ . We found that riboSNitches were more likely to be pathogenic. Moreover, we predicted several UTRs and lncRNAs (long non-coding RNA) that significantly enriched or depleted riboSNitches in cancer genomes, indicative of potential cancer driver or essential noncoding elements. Our study highlights the possibly neglected importance of RNA secondary structure in cancer genomes and provides a new strategy to identify new cancer-associated genes.

Background Previous studies suggested that non-invasive preimplantation genetic testing (niPGT) for intracytoplasmic sperm injection (ICSI) blastocysts can be used to identify chromosomal ploidy and chromosomal abnormalities. Here, we report the feasibility and performance of niPGT for conventional in vitro fertilization (IVF) blastocysts. Methods This was a prospective observational study. In the preclinical stage, whole genome amplification and NGS were performed using the sperm spent culture medium (SCM). Then, trophectoderm (TE) biopsies and corresponding SCM derived from 27 conventional IVF monopronuclear embryos were collected. In the clinical stage, samples from 25 conventional IVF cycles and 37 ICSI cycles from April 2020–August 2021 were collected for performance evaluation. Results Preclinically, we confirmed failed sperm DNA amplification under the current amplification system. Subsequent niPGT from the 27 monopronuclear blastocysts showed 69.2% concordance with PGT results of corresponding TE biopsies. In the clinical stage, no paternal contamination was observed in any of the 161 SCM samples from conventional IVF. While maternal contamination was observed in 29.8% (48/161) SCM samples, only 2.5% (4/161) samples had a contamination ratio ≥ 50%. Compared with that of TE biopsy, the performances of NiPGT from 161 conventional IVF embryos and 122 ICSI embryos were not significantly different (P > 0.05), with ploidy concordance rates of 75% and 74.6% for IVF and ICSI methods, respectively. Finally, evaluation of the euploid probability of embryos with different types of niPGT results showed prediction probabilities of 82.8%, 77.8%, 62.5%, 50.0%, 40.9% and 18.4% for euploidy, sex-chromosome mosaics only, low-level mosaics, multiple abnormal chromosomes, high-level mosaics and aneuploidy, respectively. Conclusions Our research results preliminarily confirm that the niPGT approach using SCM from conventional IVF has comparable performance with ICSI and might broadening the application scope of niPGT.

Background Embryo implantation in a receptive endometrium is crucial for successful pregnancy. Endometrial receptivity (ER) prediction tools based on endometrial transcriptome biomarkers by endometrial biopsy have been used to guide successful embryo implantation in in vitro fertilization (IVF) patients. However, no reliable noninvasive ER prediction method has been established, and one is greatly needed. We aimed to identify biomarkers from uterine fluid transcriptomic sequencing data for establishing noninvasive ER prediction tool and to evaluate its clinical application potential in patients undergoing IVF. Methods The non-invasive RNA-seq based endometrial receptivity test (nirsERT) was established by analyzing transcriptomic profile of 144 uterine fluid specimens (LH + 5, LH + 7, and LH + 9) at three different receptive status from 48 IVF patients with normal ER in combination with random forest algorithm. Subsequently, 22 IVF patients who underwent frozen-thaw blastocyst transfer were recruited and analyzed the correlation between the predicted results of nirsERT and pregnancy outcomes. Results A total of 864 ER-associated differentially expressed genes (DEGs) involved in biological processes associated with endometrium-embryo crosstalk, including protein binding, signal reception and transduction, biomacromolecule transport and cell-cell adherens junctions, were selected. Subsequently, a nirsERT model consisting of 87 markers and 3 hub genes was established using a random forest algorithm. 10-fold cross-validation resulted in a mean accuracy of 93.0%. A small cohort ( n  = 22) retrospective observation shows that 77.8% (14/18) of IVF patients predicted with a normal WOI had successful intrauterine pregnancies, while none of the 3 patients with a displaced WOI had successful pregnancies. One patient failed due to poor sequencing data quality. Conclusions NirsERT based on uterine fluid transcriptome biomarkers can predict the WOI period relatively accurately and may serve as a noninvasive, reliable and same cycle test for ER in reproductive clinics. Trial registration Chinese Clinical Trial Registry: ChiCTR-DDD-17013375. Registered 14 November 2017, http://www.chictr.org.cn/index.aspx .

Background The objective of this study was to explore the clinical application of noninvasive chromosomal screening (NICS) for elective single-blastocyst transfer (eSBT) in frozen-thawed cycles. Methods This study retrospectively analysed the data of 212 frozen-thawed single-blastocyst transfers performed in our centre from January 2019 to July 2019. The frozen embryos were selected based on morphological grades and placed in preincubation for 6 h after warming. Then spent microdroplet culture media of frozen-thawed blastocysts were harvested and subjected to NICS. The clinical outcomes were evaluated and further stratified analysis were performed, especially different fertilization approaches. Results The clinical pregnancy, ongoing pregnancy, and live birth rates in the euploidy group were significantly higher than those in the aneuploidy group (56.2% versus 29.4%) but were nonsignificantly different from those in the chaotic abnormal/NA embryos group (56.2% versus 60.4%). Compared with day6 (D6) blastocysts, D5 blastocysts had a nonsignificantly different euploidy rate (40.4% versus 48.1%, P  = 0.320) but significantly increased clinical pregnancy (57.7% versus 22.2%, P  < 0.001), ongoing pregnancy (48.1% versus 14.8%, P  < 0.001), and live birth rates (48.1% versus 13.0%, P  < 0.001). The percentage of chaotic abnormal/NA embryos group was significantly higher among D5 embryos than among D6 embryos (30.1% versus 11.1%, P  = 0.006). The percentage of aneuploid embryos was higher among the embryos with lower morphological quality(21.5% among ‘good’ embryos versus 34.6% among ‘fair’ embryos versus 46.0% among ‘poor’ embryos, P  = 0.013); correspondingly, the overall clinical pregnancy, ongoing pregnancy and live birth rate rates showed similar declines. Conclusions NICS combined with morphological assessment is an effective tool to guide frozen-thawed SBT. The optimal embryo for SBT is a ‘euploid embryo with good morphology’, followed sequentially by a ‘chaotic abnormal/NA embryo with good morphology’, ‘euploid embryo with fair morphology’, and ‘chaotic abnormal/NA embryo with fair morphology’.

RNA sequencing (RNA-Seq) technology provides the detailed transcriptomic information for a biological sample. Using the RNA-Seq data of six organs from nine vertebrate species, we identified a number of organ-specifically expressed or repressed orthologous genes whose expression patterns are mostly conserved across nine species. Our analyses show the following results: (i) About 80% of these genes have a chordate or more ancient origin and more than half of them are the legacy of one or multiple rounds of large-scale gene duplication events. (ii) Their evolutionary rates are shaped by the organ in which they are expressed or repressed, e.g. the genes specially expressed in testis and liver generally evolve more than twice as fast as the ones specially expressed in brain and cerebellum. The organ-specific transcription factors were discriminated from these genes. The ChIP-seq data from the ENCODE project also revealed the transcription-related factors that might be involved in regulating human organ-specifically expressed or repressed genes. Some of them are shared by all six human organs. The comparison of ENCODE data with mouse/chicken ChIP-seq data proposes that organ-specifically expressed or repressed orthologous genes are regulated in various combinatorial fashions in different species, although their expression features are conserved among these species. We found that the duplication events in some gene families might help explain the quick organ/tissue divergence in vertebrate lineage. The phylogenetic analysis of testis-specifically expressed genes suggests that some of them are prone to develop new functions for other organs/tissues.

Background Gene duplication and subsequent functional divergence especially expression divergence have been widely considered as main sources for evolutionary innovations. Many studies evidenced that genetic regulatory network evolved rapidly shortly after gene duplication, thus leading to accelerated expression divergence and diversification. However, little is known whether epigenetic factors have mediated the evolution of expression regulation since gene duplication. In this study, we conducted detailed analyses on yeast histone modification (HM), the major epigenetics type in this organism, as well as other available functional genomics data to address this issue. Results Duplicate genes, on average, share more common HM-code patterns than random singleton pairs in their promoters and open reading frames (ORF). Though HM-code divergence between duplicates in both promoter and ORF regions increase with their sequence divergence, the HM-code in ORF region evolves slower than that in promoter region, probably owing to the functional constraints imposed on protein sequences. After excluding the confounding effect of sequence divergence (or evolutionary time), we found the evidence supporting the notion that in yeast, the HM-code may co-evolve with cis - and trans -regulatory factors. Moreover, we observed that deletion of some yeast HM-related enzymes increases the expression divergence between duplicate genes, yet the effect is lower than the case of transcription factor (TF) deletion or environmental stresses. Conclusions Our analyses demonstrate that after gene duplication, yeast histone modification profile between duplicates diverged with evolutionary time, similar to genetic regulatory elements. Moreover, we found the evidence of the co-evolution between genetic and epigenetic elements since gene duplication, together contributing to the expression divergence between duplicate genes.