Quantitative and Qualitative QC of Next-Generation Sequencing for Detecting Somatic Variants: An Example of Detecting Clonal Hematopoiesis of Indeterminate Potential.

BACKGROUND: Because next-generation sequencing (NGS) for detecting somatic mutations has been adopted in clinical fields, both qualitative and quantitative QC of the somatic variants through whole coding regions detected by NGS is crucial. However, specific applications or guidelines, especially for quantitative QC, are currently insufficient. Our goal was to devise a practical approach for both quantitative and qualitative QC using an example of detecting clonal hematopoiesis of indeterminate potential (CHIP). METHODS: We applied the QC scheme using commercial reference materials and in-house QC materials (IQCM) composed of haplotype map and cancer cell lines for monitoring CHIP. RESULTS: This approach efficiently validated a customized CHIP NGS assay. Accuracy, analytical sensitivity, analytical specificity, qualitative precision (concordance), and limit of detection achieved were 99.87%, 98.53%, 100.00%, 100.00%, and 1.00%, respectively. The quantitative precision analysis also had a higher CV percentage at a lower alternative read depth (R2 = 0.749∼0.858). Use of IQCM ensured more than 100-fold reduction in the cost per run compared with that achieved using commercial reference materials. CONCLUSION: Our approach determined the general analytical performance of NGS for detecting CHIP and recognized limitations such as lower precision at a lower level of variant burden. This approach could also be theoretically expanded to a general NGS assay for detecting somatic variants. Considering the reliable NGS results and cost-effectiveness, we propose the use of IQCM for QC of NGS assays at clinical laboratories.

DeviCNV: detection and visualization of exon-level copy number variants in targeted next-generation sequencing data.

BACKGROUND: Targeted next-generation sequencing (NGS) is increasingly being adopted in clinical laboratories for genomic diagnostic tests. RESULTS: We developed a new computational method, DeviCNV, intended for the detection of exon-level copy number variants (CNVs) in targeted NGS data. DeviCNV builds linear regression models with bootstrapping for every probe to capture the relationship between read depth of an individual probe and the median of read depth values of all probes in the sample. From the regression models, it estimates the read depth ratio of the observed and predicted read depth with confidence interval for each probe which is applied to a circular binary segmentation (CBS) algorithm to obtain CNV candidates. Then, it assigns confidence scores to those candidates based on the reliability and strength of the CNV signals inferred from the read depth ratios of the probes within them. Finally, it also provides gene-centric plots with confidence levels of CNV candidates for visual inspection. We applied DeviCNV to targeted NGS data generated for newborn screening and demonstrated its ability to detect novel pathogenic CNVs from clinical samples. CONCLUSIONS: We propose a new pragmatic method for detecting CNVs in targeted NGS data with an intuitive visualization and a systematic method to assign confidence scores for candidate CNVs. Since DeviCNV was developed for use in clinical diagnosis, sensitivity is increased by the detection of exon-level CNVs.

Mutations in DDX58, which encodes RIG-I, cause atypical Singleton-Merten syndrome.

Singleton-Merten syndrome (SMS) is an autosomal-dominant multi-system disorder characterized by dental dysplasia, aortic calcification, skeletal abnormalities, glaucoma, psoriasis, and other conditions. Despite an apparent autosomal-dominant pattern of inheritance, the genetic background of SMS and information about its phenotypic heterogeneity remain unknown. Recently, we found a family affected by glaucoma, aortic calcification, and skeletal abnormalities. Unlike subjects with classic SMS, affected individuals showed normal dentition, suggesting atypical SMS. To identify genetic causes of the disease, we performed exome sequencing in this family and identified a variant (c.1118A>C [p.Glu373Ala]) of DDX58, whose protein product is also known as RIG-I. Further analysis of DDX58 in 100 individuals with congenital glaucoma identified another variant (c.803G>T [p.Cys268Phe]) in a family who harbored neither dental anomalies nor aortic calcification but who suffered from glaucoma and skeletal abnormalities. Cys268 and Glu373 residues of DDX58 belong to ATP-binding motifs I and II, respectively, and these residues are predicted to be located closer to the ADP and RNA molecules than other nonpathogenic missense variants by protein structure analysis. Functional assays revealed that DDX58 alterations confer constitutive activation and thus lead to increased interferon (IFN) activity and IFN-stimulated gene expression. In addition, when we transduced primary human trabecular meshwork cells with c.803G>T (p.Cys268Phe) and c.1118A>C (p.Glu373Ala) mutants, cytopathic effects and a significant decrease in cell number were observed. Taken together, our results demonstrate that DDX58 mutations cause atypical SMS manifesting with variable expression of glaucoma, aortic calcification, and skeletal abnormalities without dental anomalies.

CACG: a database for comparative analysis of conjoined genes.

A conjoined gene is defined as one formed at the time of transcription by combining at least part of one exon from each of two or more distinct genes that lie on the same chromosome, in the same or opposite orientation, which translate independently into different proteins. We comparatively studied the extent of conjoined genes in thirteen genomes by analyzing the public databases of expressed sequence tags and mRNA sequences using a set of computational tools designed to identify conjoined genes on the same DNA strand or opposite DNA strands of the same genomic locus. The CACG database, available at http://cgc.kribb.re.kr/map/, includes a number of conjoined genes (7131-human, 2-chimpanzee, 5-orangutan, 57-chicken, 4-rhesus monkey, 651-cow, 27-dog, 2512-mouse, 263-rat, 1482-zebrafish, 5-horse, 29-sheep, and 8-medaka) and is very effective and easy to use to analyze the evolutionary process of conjoined genes when comparing different species.

Genome sequence of Lactobacillus fructivorans KCTC 3543.

Lactobacillus fructivorans is important in the generation of particular flavors and in other ripening processes associated with fermented food. Here, we present the draft genome sequence of the type strain Lactobacillus fructivorans KCTC 3543 (1,373,326 bp, with a G+C content of 38.9%), which consists of 5 scaffolds. The genome sequence was obtained by using a whole-genome shotgun strategy with Roche 454 GS (FLX Titanium) pyrosequencing, and all of the reads were assembled using Newbler Assembler 2.3.

Genome sequence of Peptoniphilus rhinitidis 1-13T, an anaerobic coccus strain isolated from clinical specimens.

A new Peptoniphilus species has been isolated from samples from a patient who was scheduled for endoscopic sinus surgery for chronic rhinosinusitis. The isolate, Peptoniphilus rhinitidis 1-13(T) (KCTC 5985(T)), can use peptone as a sole carbon source and produce butyrate as a metabolic end product. This is the first report of the draft genome sequence of a novel species in the genus Peptoniphilus within the group of Gram-positive anaerobic cocci.

Genome sequence of Myroides injenensis M09-0166(T), isolated from clinical specimens.

A new Myroides species has been isolated from the urine of a patient with fever in spite of multiple antibiotic treatments who had undergone a radical hysterectomy for cervical cancer and percutaneous nephrostomies for hydronephrosis in the past. The isolate, Myroides injenensis M09-0166(T) (KCTC 23367(T)), showed a high level of resistance to multiple antibiotic agents. Here we provide the first report of the draft genome sequence of a novel species in the genus Myroides within the nonfermenting Gram-negative group.

Genome sequence of the anaerobic bacterium Clostridium arbusti SL206(T).

A new Clostridium species has been isolated from pear orchard soil in Daejeon, Republic of Korea. The isolate, Clostridium arbusti SL206(T) (KCTC 5449(T)), showed a nitrogenase activity as well as an organic acid production. Here we first report the draft genome sequence of a novel species in the genus Clostridium within the largest Gram-positive group.

Novel mechanism of conjoined gene formation in the human genome.

Recently, conjoined genes (CGs) have emerged as important genetic factors necessary for understanding the human genome. However, their formation mechanism and precise structures have remained mysterious. Based on a detailed structural analysis of 57 human CG transcript variants (CGTVs, discovered in this study) and all (833) known CGs in the human genome, we discovered that the poly(A) signal site from the upstream parent gene region is completely removed via the skipping or truncation of the final exon; consequently, CG transcription is terminated at the poly(A) signal site of the downstream parent gene. This result led us to propose a novel mechanism of CG formation: the complete removal of the poly(A) signal site from the upstream parent gene is a prerequisite for the CG transcriptional machinery to continue transcribing uninterrupted into the intergenic region and downstream parent gene. The removal of the poly(A) signal sequence from the upstream gene region appears to be caused by a deletion or truncation mutation in the human genome rather than post-transcriptional trans-splicing events. With respect to the characteristics of CG sequence structures, we found that intergenic regions are hot spots for novel exon creation during CGTV formation and that exons farther from the intergenic regions are more highly conserved in the CGTVs. Interestingly, many novel exons newly created within the intergenic and intragenic regions originated from transposable element sequences. Additionally, the CGTVs showed tumor tissue-biased expression. In conclusion, our study provides novel insights into the CG formation mechanism and expands the present concepts of the genetic structural landscape, gene regulation, and gene formation mechanisms in the human genome.

Genome Sequence of Leuconostoc gelidum KCTC 3527, Isolated from Kimchi.

Leuconostoc gelidum KCTC 3527 is found mainly in vegetables and plays an important role in vegetable fermentation, including that of Korean traditional kimchi. Here we announce the draft genome sequence of Leuconostoc gelidum KCTC 3527, isolated from Korean traditional kimchi, and describe major findings from its annotation.

Genome sequence of Lactobacillus coryniformis subsp. coryniformis KCTC 3167.

Lactobacillus coryniformis subsp. coryniformis is known to be present during the manufacturing process of kimchi, the best-known traditional Korean dish. Here, we present the draft genome sequence of Lactobacillus coryniformis subsp. coryniformis type strain KCTC 3167 (2,964,752 bp, with a G+C content of 42.8%), which consists of 55 scaffolds.

Genome sequence of Leuconostoc fallax KCTC 3537.

Leuconostoc fallax is known to be present during the manufacturing process of kimchi, the best-known traditional Korean dish. Here, we present the draft genome sequence of the type strain Leuconostoc fallax KCTC 3537 (1,638,971 bp, with a G+C content of 37.5%), which consists of 30 large contigs (>100 bp in size).

Genome Sequence of Weissella cibaria KACC 11862.

Weissella cibaria KACC 11862 is a Gram-positive, heterofermentative, Leuconostoc-like lactic acid bacterium that is widely distributed in Korean traditional foods such as kimchi. Here we report the draft genome sequence of the type strain, W. cibaria KACC 11862 (1,599 known genes, 80 RNA genes), which consists of 72 large contigs (>100 bp in size).

Genome sequence of Leuconostoc pseudomesenteroides KCTC 3652.

We announce the genome sequence of one of the most prevalent lactic acid bacteria present during the manufacturing process of cane juice, the type strain Leuconostoc pseudomesenteroides KCTC 3652 (3,244,985 bp, with a G+C content of 38.3%), which consists of 1,160 large contigs (>100 bp in size). All of the contigs were assembled by the Newbler Assembler 2.3 software program (454 Life Sciences).

Genome sequence of Lactobacillus cypricasei KCTC 13900.

Lactobacillus cypricasei KCTC 13900 is important in the generation of particular flavors and in other ripening processes associated with specific cheeses. Here, we announce the draft genome sequence of Lactobacillus cypricasei KCTC 13900, isolated from cheeses, and describe major findings from its annotation.

Draft genome sequence of Lactobacillus zeae KCTC 3804.

We announce the draft genome sequence of the type strain Lactobacillus zeae KCTC 3804 (3,110,326 bp, with a G+C content of 47.8%), which is one of the most prevalent lactic acid bacteria present during the processing of raw cow's milk. The genome consists of 113 large contigs (>100 bp). All of the contigs were assembled by Newbler Assembler 2.3 (454 Life Science).

Draft genome sequence of Lactobacillus mali KCTC 3596.

We announce the draft genome sequence of the type strain Lactobacillus mali KCTC 3596 (2,652,969 bp, with a G+C content of 36.0%), which is one of the most prevalent lactic acid bacteria present during the manufacturing process of apple juice. The genome consists of 122 large contigs (>100 bp). All of the contigs were assembled by Newbler Assembler 2.3 (454 Life Science).

Genome sequence of Lactobacillus suebicus KCTC 3549.

Lactobacillus suebicus is important in the generation of particular flavors and in other ripening processes associated with apple mash. Here, we present the draft genome sequence of the type strain Lactobacillus suebicus KCTC 3549 (2,656,936 bp, with a G+C content of 39.0%), which consists of 143 large contigs (>100 bp).

Draft genome sequence of Lactobacillus malefermentans KCTC 3548.

We announce the draft genome sequence of the type strain Lactobacillus malefermentans KCTC 3548 (2,003,922 bp, with a G+C content of 41.1%), which is one of the most prevalent lactic acid bacteria present during the manufacturing process of beer; the genome consists of 172 large contigs (>100 bp in size). All of the contigs were assembled by using Newbler Assembler 2.3 (454 Life Science).

Genome sequence of Lactobacillus versmoldensis KCTC 3814.

Lactobacillus versmoldensis KCTC 3814 was isolated from raw fermented poultry salami. The species was present in high numbers and frequently dominated the lactic acid bacteria (LAB) populations of the products. Here, we announce the draft genome sequence of Lactobacillus versmoldensis KCTC 3814, isolated from poultry salami, and describe major findings from its annotation.