BackgroundTo take advantage of affordable high-throughput next-generation sequencing technologies to characterize microbial community composition often requires the development of improved methods to overcome technical limitations inherent to the sequencing platforms. Sequencing low sequence diversity libraries such as 16S rRNA amplicons has been problematic on the Illumina MiSeq platform and often generates sequences of suboptimal quality.ResultsHere we present an improved dual-indexing amplification and sequencing approach to assess the composition of microbial communities from clinical samples using the V3-V4 region of the 16S rRNA gene on the Illumina MiSeq platform. We introduced a 0 to 7 bp “heterogeneity spacer” to the index sequence that allows an equal proportion of samples to be sequenced out of phase.ConclusionsOur approach yields high quality sequence data from 16S rRNA gene amplicons using both 250 bp and 300 bp paired-end MiSeq protocols and provides a flexible and cost-effective sequencing option.

Apolipoprotein C-III (apoC-III) inhibits triglyceride hydrolysis and has been implicated in coronary artery disease. Through a genome-wide association study, we have found that about 5% of the Lancaster Amish are heterozygous carriers of a null mutation (R19X) in the gene encoding apoC-III (APOC3) and, as a result, express half the amount of apoC-III present in noncarriers. Mutation carriers compared to noncarriers had lower fasting and postprandial serum triglycerides, higher levels of HDL-cholesterol and lower levels of LDL-cholesterol. Subclinical atherosclerosis, as measured by coronary artery calcification, was less common in carriers than noncarriers, suggesting that lifelong deficiency of apoC-III has a cardioprotective effect.Elevated plasma levels of low density lipoprotein cholesterol (LDL-C) and triglycerides (TG) are important contributors to premature coronary heart disease (CHD) (1-3), and genetic variants causing low LDL-C are associated with reduced risk of CHD (4). Recently, nonfasting TG was found to be an independent CHD risk factor (5,6), in one study showing higher predictive power than fasting TG (FTG), the traditional measure, likely because of the atherogenic remnant lipoproteins generated during absorption and clearance of dietary fat (5).To identify genetic factors contributing to FTG and post-prandial TG (ppTG) dietary response, we performed a single high fat feeding intervention and genome-wide association study (GWAS) in 809 Old Order Amish individuals as part of the Heredity and Phenotype Intervention (HAPI) Heart Study (7). Characteristics of these participants are shown in Table S1. These individuals were fed a milkshake containing 782 kcal/m 2 body surface area with 77.6% of these calories from fat and had blood drawn for lipid levels 0, 1, 2, 3, 4 and 6 hours after the intervention. The Affymetrix GeneChip® Human Mapping 500K Array Set was used for genotyping leukocyte DNA from these 809 participants. Traits were normalized and * This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/cgi/content/full/322/5908/1702. Their manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the Copyright Act without the prior, written permission of AAAS. analyses accounting for sex and sex-specific age and age 2 , body mass index (BMI) and relatedness among participants were performed as described in the Methods (8).Results of the GWAS of FTG and ppTG (as estimated by the incremental area under the curve, iAUCTG (8)), transformed by their natural logarithm (ln), are shown in Table S2 and Figure S1. The strongest evidence for association with both ln-FTG (p = 3.8 × 10 −14 ) and ln-iAUCTG (p = 2.8 × 10 −10 ) occurred on chromosome 11q23 at single nucleotide polymorphism (SNP) rs10892151, which had a minor allele frequency (MAF) of 0.028 (A allele; Table S2). SNP rs10892151 is located within an intron of th...