Structural variant analysis for linked-read sequencing data with gemtools.

SUMMARY: Linked-read sequencing generates synthetic long reads which are useful for the detection and analysis of structural variants (SVs). The software associated with 10× Genomics linked-read sequencing, Long Ranger, generates the essential output files (BAM, VCF, SV BEDPE) necessary for downstream analyses. However, to perform downstream analyses requires the user to customize their own tools to handle the unique features of linked-read sequencing data. Here, we describe gemtools, a collection of tools for the downstream and in-depth analysis of SVs from linked-read data. Gemtools uses the barcoded aligned reads and the Megabase-scale phase blocks to determine haplotypes of SV breakpoints and delineate complex breakpoint configurations at the resolution of single DNA molecules. The gemtools package is a suite of tools that provides the user with the flexibility to perform basic functions on their linked-read sequencing output in order to address even more questions. AVAILABILITY AND IMPLEMENTATION: The gemtools package is freely available for download at: https://github.com/sgreer77/gemtools. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

[Exogenous CRX gene induces Müller cell-derived progenitors to differentiate into photoreceptors].

Objective: To investigate whether exogenous CRX gene would be able to induce Müller cells-derived progenitors to differentiate into photoreceptors. Methods: Experimental study. Müller cells-derived progenitors resulted from primary Müller cells isolated from KunMing mice(5-7 days old) and cultured in free-serum media. Markers of Müller cells(glutamine synthetase, GS and Vimentin) and stem cells (Nestin and Sox2) were analysed by immnocytochemical assays. The secondary passage progenitors were divided into three groups: (1)the control group; (2)the empty vector group was transfected with lentivirus GFP; (3)the treated group was transfected with lentivirus GFP-CRX. After differentiation for 7 days, 7 days after differentiation, the expression of markers of photoreceptors were analyzed by q-PCR and Western blot assay. Results: There were 96.03%±1.21% of Müllerz cells cultured in vitro were immunoreactive to both GS and Vimentin. The dedifferentiation cells expressed Nestin and Sox2. After 7 days of induction, Exogenous CRX induced Müller cell-derived progenitors to differentiate into rod-like cells showed appearance like neuron morphology. q-PCR demonstrated that mRNAs of CRX and Rhodopsin were upregulated greatly. CRX mRNA were 9 times (P<0.05) and Rhodopsin mRNA were 20 times (P<0.05). The difference between the control group and the empty vector group was not statistically significant. Western blot showed that the expression of CRX was upregulated significantly, and was 2.7 times(P<0.05). But expression of Rhodopsin was weak and was nearly not detected in the control group and empty vector group. The expression of S-opsin was not detected. Conclusion: CRX gene could induce the differentiation of Müller cell-derived progenitor into rod photoreceptors, indicating a new avenue to study müller cells as endogenous seed cells for retinal photoreceptor. (Chin J Ophthalmol, 2018, 54: 923-928).

A functional assay for mutations in tumor suppressor genes caused by mismatch repair deficiency.

The coding sequences of multiple human tumor suppressor genes include microsatellite sequences that are prone to mutations. Saccharomyces cerevisiae strains deficient in DNA mismatch repair (MMR) can be used to determine de novo mutation rates of these human tumor suppressor genes as well as any other gene sequence. Microsatellites in human TGFBR2, PTEN and APC genes were placed in yeast vectors and analyzed in isogenic yeast strains that were wild-type or deletion mutants for MSH2 or MLH1. In MMR-deficient strains, the vector containing the (A)(10) microsatellite sequence of TGFBR2 had a mutation rate (mutations/cell division) of 1.4 x 10(-4), compared to a mutation rate of 1.7 x 10(-6) in the wild-type strain. In MMR-deficient strains, mutation rates in PTEN and APC were also elevated above background levels. PTEN mutation rates were higher in both msh2 (4.4 x 10-5) and mlh1 strains (2.3 x 10-5). APC mutation rates in the msh2 strain (2.4 x 10-6) and the mlh1 strain (1.7 x 10-6) were also significantly, but less dramatically, elevated over background. Mutations selected for in the yeast screen were identical to those previously observed in human tumor samples with microsatellite instability (MSI). This functional assay has applicability in providing quantitative data about microsatellite mutation rates caused by MMR deficiency in any human tumor suppressor gene sequence. It can also be applied as a genetic screen to identify new genes that are vulnerable to such microsatellite mutations and thus may be involved in the neoplastic development of tumors with MSI.

Spondyloepimetaphyseal dysplasia with joint laxity (SEMDJL): presentation in two unrelated patients in the United States.

This is a report of two North American patients with spondyloepimetaphyseal dysplasia with joint laxity, an uncommon autosomal recessive skeletal dysplasia rarely reported outside of South Africa. Patients with SEMDJL have vertebral abnormalities and ligamentous laxity that results in spinal misalignment and progressive severe kyphoscoliosis, thoracic asymmetry, and respiratory compromise resulting in early death. Nonaxial skeletal involvement includes elbow deformities with radial head dislocation, dislocated hips, clubbed feet, and tapered fingers with spatulate distal phalanges. Many affected children have an oval face, flat midface, prominent eyes with blue sclerae, and a long philtrum. Palatal abnormalities and congenital heart disease are also observed. Diagnosis in infancy may be difficult because many of the typical findings are not apparent early and only evolve over time. We review the physical and radiographic findings in two unrelated patients with this disorder in order to increase the awareness of this disorder, particularly for clinicians outside of South Africa.

Preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins.

AIM: To prepare hybridoma cell lines that secrete monoclonal antibodies against hepatitis C virus (HCV) recombinant proteins NS3 and NS5 and to evaluate their use in the study of HCV NS3 and NS5 antigen distribution in human liver tissue. METHODS: Hybridoma cell lines were generated using spleen cells from BALB/C mice immunized with recombinant NS3 and NS5 proteins, following conventional protocols. Antibody-secreting cells were screened by solid phase ELISA and cloned by limited dilution. The specificity of the monoclonal antibodies was determined by testing hybridoma culture supernatants by Western blots of E. coli expressing the recombinant HCV proteins and ELISA with HCV core and hepatitis B virus (HBV) antigens. The monoclonal antibodies were employed in immunohistochemistry studies to determine the distribution of HCV NS5 and NS3 antigens in 51 paraffin embedded human liver tissue samples. RESULTS: Eight hybridoma cell lines secreting monoclonal antibodies against HCV NS3 and NS5 proteins were generated and named 2B6, 2F3, 3D8, 3D9, 8B2, 6F11, 4C6 and 7D9. Only one of them, 2B6 (secreting antibodies against NS3 protein), cross-reacted with the C7 polypeptide, a different recombinant NS3 polypeptide. The rest of the cell lines showed no cross-reactivity with HCV core or HBV antigens. In addition, monoclonal antibodies against NS3 antigens did not cross-react with NS5 antigens, and vice versa. In immunohistochemistry studies, these monoclonal antibodies did not detect HCV antigens in specimens from patients infected only with HBV (n = 20). In HCV-infected specimens (n = 31), the rates of positive detection of NS3 and NS5 antigens were 51.6% (16/31) and 54.9% (17/31), respectively. Six of these 31 specimens were from patients infected only with HCV and half of them were positive for HCV NS3 and NS5 antigens. In specimens from patients co-infected with HBV and HCV (n = 25), the rates of NS3 and NS5 antigen positive detection were 52% (13/25) and 56% (14/25), respectively, which are similar to those obtained in samples from patients infected only with HCV. In specimens from chronic active cirrhosis patients, the rates of HCV NS3 and NS5 antigen detection were 70.6% (12/17) and 76.5% (13/17), respectively. CONCLUSION: We successfully prepared monoclonal antibodies that are specific against recombinant HCV NS3 and NS5 proteins and could be useful for clinical immunohistochemistry diagnosis.