Evaluation of Hybridization Capture Versus Amplicon‐Based Methods for Whole‐Exome Sequencing

Next‐generation sequencing has aided characterization of genomic variation. While whole‐genome sequencing may capture all possible mutations, whole‐exome sequencing remains cost‐effective and captures most phenotype‐altering mutations. Initial strategies for exome enrichment utilized a hybridization‐based capture approach. Recently, amplicon‐based methods were designed to simplify preparation and utilize smaller DNA inputs. We evaluated two hybridization capture‐based and two amplicon‐based whole‐exome sequencing approaches, utilizing both Illumina and Ion Torrent sequencers, comparing on‐target alignment, uniformity, and variant calling. While the amplicon methods had higher on‐target rates, the hybridization capture‐based approaches demonstrated better uniformity. All methods identified many of the same single‐nucleotide variants, but each amplicon‐based method missed variants detected by the other three methods and reported additional variants discordant with all three other technologies. Many of these potential false positives or negatives appear to result from limited coverage, low variant frequency, vicinity to read starts/ends, or the need for platform‐specific variant calling algorithms. All methods demonstrated effective copy‐number variant calling when evaluated against a single‐nucleotide polymorphism array. This study illustrates some differences between whole‐exome sequencing approaches, highlights the need for selecting appropriate variant calling based on capture method, and will aid laboratories in selecting their preferred approach.     

Early Childhood Gut Microbiomes Show Strong Geographic Differences Among Subjects at High Risk for Type 1 Diabetes

OBJECTIVE

Gut microbiome dysbiosis is associated with numerous diseases, including type 1 diabetes. This pilot study determines how geographical location affects the microbiome of infants at high risk for type 1 diabetes in a population of homogenous HLA class II genotypes.

RESEARCH DESIGN AND METHODS

High-throughput 16S rRNA sequencing was performed on stool samples collected from 90 high-risk, nonautoimmune infants participating in The Environmental Determinants of Diabetes in the Young (TEDDY) study in the U.S., Germany, Sweden, and Finland.

RESULTS

Study site–specific patterns of gut colonization share characteristics across continents. Finland and Colorado have a significantly lower bacterial diversity, while Sweden and Washington state are dominated by Bifidobacterium in early life. Bacterial community diversity over time is significantly different by geographical location.

CONCLUSIONS

The microbiome of high-risk infants is associated with geographical location. Future studies aiming to identify the microbiome disease phenotype need to carefully consider the geographical origin of subjects.     

Shc proteins influence the activities of enzymes involved in fatty acid oxidation and ketogenesis

ObjectivesShcKO mice have low body fat and resist weight gain on a high fat diet, indicating that Shc proteins may influence enzymes involved in β-oxidation. To investigate this idea, the activities of β-oxidation and ketone body metabolism enzymes were measured.MethodsThe activities of β-oxidation enzymes (acyl-CoA dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase and ketoacyl-CoA thiolase) in liver and hindlimb skeletal muscle, ketolytic enzymes (acetoacetyl-CoA thiolase, β-hydroxybutyrate dehydrogenase and 3-oxoacid-CoA transferase) in skeletal muscle, and ketogenic enzymes (acetoacetyl-CoA thiolase and β-hydroxybutyrate dehydrogenase) in liver were measured from wild-type and ShcKO mice.ResultsThe activities of β-oxidation enzymes were increased (P < .05) in the ShcKO compared to wild-type mice in the fasted but not the fed state. In contrast, no uniform increases in the ketolytic enzyme activities were observed between ShcKO and wild-type mice. In liver, the activities of ketogenic enzymes were increased (P < .05) in ShcKO compared to wild-type mice in both the fed and fasted states. Levels of phosphorylated hormone sensitive lipase from adipocytes were also increased (P < .05) in fasted ShcKO mice.ConclusionThese studies indicate that the low Shc levels in ShcKO mice result in increased liver and muscle β-oxidation enzyme activities in response to fasting and induce chronic increases in the activity of liver ketogenic enzymes. Decreases in the level of Shc proteins should be considered as possible contributors to the increase in activity of fatty acid oxidation enzymes in response to physiological conditions which increase reliance on fatty acids as a source of energy.     

Laparoscopic cholecystectomy is the preferred approach in cirrhosis: a nationwide,population-based study

Background/aim

To assess the impact of open versus laparoscopic surgery in cirrhotic patients undergoing a cholecystectomy using the Nationwide Inpatient Sample (NIS).

Methods

All patients with cirrhosis who underwent a cholecystectomy (open or laparoscopic) between 2003 and 2006 were queried from the NIS. Associated complications including infection, transfusion, reoperation, liver failure and mortality were determined.

Results

A total of 3240 patients with cirrhosis underwent a cholecystectomy: 383 patients underwent an open cholecystectomy (OC) whereas 2857 patients underwent a laparoscopic cholecystectomy (LC), which included 412 patients converted (LCC) from a LC to an OC. Post-operative infection was higher in OC as opposed to a laparoscopic cholecystectomy (TLC) or LCC (3.5% versus 0.7% versus 0.2%, P < 0.0001). The need for a blood transfusion was significantly higher in the OC and LCC groups as compared with the TLC group (19.2% versus 14.4% versus 6.2%, P < 0.0001). Reoperation was more frequent after OC or LCC versus TLC (1.5% versus 2.5% versus 0.8%, P = 0.007). In-hospital mortality was higher after OC as compared with TLC and LCC (8.3% versus 1.3% versus 1.4%, P < 0.0001).

Conclusion

Patients with cirrhosis have increased in-hospital morbidity and mortality after an open as opposed to a laparoscopic or conversion to an open cholecystectomy. LC should be the preferred initial approach in cirrhotic patients.     

Late-infantile Batten disease: Purification of the subunit c of the mitochondrial ATP synthase from storage material

The accumulation of subunit c of the mitochondrial ATP synthase in late-infantile neuronal lipofuscinosis (LINCL) and juvenile neuronal lipofuscinosis (JNCL) is well documented. The purification of the subunit from diverse sources has been reported previously, although not from the brain of Batten disease patients. This proteolipid has now been purified from late-infantile Batten disease brain. The procedures used were an original combination of the conventional solubilisation, differential centrifugation, organic solvent extractions, preparative gel electrophoresis, and FPLC. Gel filtration of the purified protein indicated molecular mass equal to or greater than 2 × 10⁶ Da; however, electrophoresis of this pure protein suggested a molecular mass of approximately 3,500 Da, which is a characteristic of subunit c. The pure protein may be solubilised in aqueous buffer containing <1% lithium dodecyl sulphate (LDS). The protein binds dicyclohexylcarbodiimide (DCCD) and shows immunoreactivity to antibodies raised against ovine storage bodies. © 1995 Wiley-Liss, Inc.