Survey of the Applications of NGS to Whole-Genome Sequencing and Expression Profiling

Recently, the technologies of DNA sequence variation and gene expression profiling have been used widely as approaches in the expertise of genome biology and genetics. The application to genome study has been particularly developed with the introduction of the next-generation DNA sequencer (NGS) Roche/454 and Illumina/Solexa systems, along with bioinformation analysis technologies of whole-genome de novo assembly, expression profiling, DNA variation discovery, and genotyping. Both massive whole-genome shotgun paired-end sequencing and mate paired-end sequencing data are important steps for constructing de novo assembly of novel genome sequencing data. It is necessary to have DNA sequence information from a multiplatform NGS with at least 2x and 30x depth sequence of genome coverage using Roche/454 and Illumina/Solexa, respectively, for effective an way of de novo assembly. Massive short-length reading data from the Illumina/Solexa system is enough to discover DNA variation, resulting in reducing the cost of DNA sequencing. Whole-genome expression profile data are useful to approach genome system biology with quantification of expressed RNAs from a whole-genome transcriptome, depending on the tissue samples. The hybrid mRNA sequences from Rohce/454 and Illumina/Solexa are more powerful to find novel genes through de novo assembly in any whole-genome sequenced species. The 20x and 50x coverage of the estimated transcriptome sequences using Roche/454 and Illumina/Solexa, respectively, is effective to create novel expressed reference sequences. However, only an average 30x coverage of a transcriptome with short read sequences of Illumina/Solexa is enough to check expression quantification, compared to the reference expressed sequence tag sequence.

Construction of Various Copy Number Plasmid Vectors and Their Utility for Genome Sequencing

We developed various plasmid cloning vectors that are useful in the construction of genomic and shotgun libraries. Two medium copy vectors, pCUGIblu21(pCb21) and pAGIblu21 (pAb21), which are resistant to kanamycin (KmR) and chloramphenicol (CamR), respectively, are useful for cloning DNA inserts ranging from 5kb to 15kb. Two high copy vectors, pCUGIblu31 (pCb31) and pAGIblu31 (pAb31), containing KmR and CamR, respectively, are useful for DNA inserts less than 5kb. These vectors are well adapted for large-scale genome sequencing projects by providing choice of copy number and selectable marker. The small vector size is another advantage of these vectors. All vectors contain lacZ including multicloning sites that originated from pBluscriptIIsk- for easy cloning and sequencing. Two medium copy vectors contain unique and rare cutting SwaI (ATTTAAAT) restriction enzyme sites for easy determination of insert size. We developed two combined vectors, pC21A31 and pC31A21, which are combinations of (pCb21 + pAb31) and (pCb31 + Ab21),respectively. These two vectors provide four choices of vectors such as KmR and medium, CamR and high, CamR and medium, and KmR and high copy vectors by restriction enzyme cutting, dephosphorylation, and gel purification. These vectors were successfully applied to high throughput shotgun sequencing of rice, tomato, and brassica BAC clones. With an example of extremely biased hydro sheared 3 kb shotgun library of a tomato BAC clone, which is originated from cytogenetically defined peri-centromeric region, we suggest the utility of an additional 10 kb library for sequence assembly of the difficult-to-assemble BAC clone.

A Case of Gas Forming Enterocolitis Associated with Kawasaki Disease / 대한소아소화기영양학회지

Kawasaki Disease, an acute systemic vasculitis of unknown etiology, is the leading cause of acquired heart disease in children in many parts of the world. It predominantly affects children under 5 years of age and has many clinical symptoms. We experienced a case of gas forming enterocolitis associated with Kawasaki Disease. Aeromonas hydrophilia was isolated from her stool culture. So,we report the case with a brief review of its literature.

A Study for Incidence of Childhood Leukemia in Kyongnam Province, Korea / 대한소아혈액종양학회지

PURPOSE: Of the cancers in childhood, leukemia is the most frequent one. For the desirable control of childhood leukemia, the basic data for the incidence has a great importance. The authors made a report about the incidence of leukemia in childhood, which analyzed the data from 126 cases in Kyongnam province, Korea, during 1991~1995. METHODS: The data were obtained from 126 new cases of childhood leukemia who had been living in the Kyongnam province and were diagnosed at the 26 university hospitals or general hospitals in the Kyongnam area and other cities from 1991 to 1995. RESULTS: The age-and-sex adjusted annual incidence rate per 100,000 population during 1991~1995 varied from 1.82 to 2.86, and cumulative annual incidence rate was 2.41 (male 2.26 and female 2.57 respectively). Male to female sex ratio was 1:1 in total cases. By the major types of childhood leukemia, the cases were composed of acute lymphocytic leukemia 70.6%, acute myelocytic leukemia 26.9% and chronic myelocytic leukemia 2.5%. The cumulative annual incidence rate per 100,000 population (crude rate) during 1991~1995 were 2.77 in Ulsan city, 2.62 in Chinju city and 2.34 in the whole area of Kyongnam province. CONCLUSION: It was concluded that the age-and-sex adjusted annual incidence rate per 100,000 of childhood in Kyongnam province was 2.41, which was lower than that in Pusan city in the same period. And, there was no significant difference of the cumulative annual incidence rate between Ulsan area and Chinju area in the same period.

Clinical and Pathologic Analysis of Thin Glomerular Basement Membrane Disease in Children

PURPOSE : Clinical manifestations and pathologic findings of thin glomerular basement membrane disease, recognized as a common underlying disease of benign, familiar and asymptomatic hematuria has not been reported systemically in Korea. We analyzed clinical and pathologic findings of patients who were diagnosed as thin glomerular basement membrane disease. METHODS : We analyzed clinical and pathologic findings of twenty-six patients who were diagnosed as thin glomerular basement membrane disease by renal biopsy among who complained asymptomatic hematuria from 1990 to 2000. RESULTS : The subjects were aged 9.4+/-3.2 (3.0-15.8) years-old at onset of hematuria, and 11.1+/-2.2 (4.7-16.3) years-old at renal biopsy. Sexual discrepancy was more common in girls (eight boys and eighteen girls). A family history of hematuria was found in 8 patients(30.7%). Major clinical manifestation on admission was microscopic hematuria according to the findings of 3cases(11.5%) of gross hematuria, 23cases(88.5%9) of microscopic hematuria, and 1case(3.8%) of proteinuria. Microscopic hematuria persisted in all cases. Kidney biopsy showed few changes by light microscopy, but IgM, C3 and fibrinogen deposit in mesangium was found by immunofluorescent microscopy in a few cases. Electron microscopic findings have revealed thinning of the glomerular basement membrane varied from 180.9+/-35.8nm. CONCLUSION : Thin glomerular basement membrane disease might be a common cause of microscopic hematuria of children and family history was revealed in about 30%. Clinical progression was good in majorities.

The Study of the Initial Presentations of Wilson Disease at Diagonosis / 대한소아소화기영양학회지

PURPOSE: To review the clinical and laboratory features of patients with Wilson disease at diagnosis. METHODS: In this retrospective study, records of all 20 patients, who were diagnosed as having Wilson disease at the Paik hospital in Busan from 1990 to 2000, were reviewed. RESULTS: Out of 20 patients, 12 pateints (60%) have hepatic presentation alone, 2 patients (10%) have neurologic presentation, 4 patients (20%) have hepatic and neuropsychiatric presentation, and one patient (5%) has hematologic presentation at diagnosis. One patient (5%) has neither symptom nor laboratory finding of Wilson disease except very low serum ceruloplasmin level and positive family history. Family screening test revealed 3 cases of Wilson disease. 12 patients were revealed to be combined with liver cirrhosis at diagnosis. CONCLUSION: Early diagnosis and treatment is very important in patients with Wilson disease. Children or adolescents who manifest symptoms of hepatitis, who has prolonged elevation of liver enzymes, and has family history of hapatitis of unknown origin, with mild hematologic or urinary abnormalities must be suspected to have Wilson disease. Also, in adolescents with extrapyramidal symptoms or other neuropsychiatric symptoms, liver function test should be done.