Metagenomic analysis of the microbial community in kefir grains.

Kefir grains as a probiotic have been subject to microbial community identification using culture-dependent and independent methods that target specific strains in the community, or that are based on limited 16S rRNA analysis. We performed whole genome shotgun pyrosequencing using two Turkish Kefir grains. Sequencing generated 3,682,455 high quality reads for a total of ∼1.6 Gbp of data assembled into 6151 contigs with a total length of ∼24 Mbp. Species identification mapped 88.16% and 93.81% of the reads rendering 4 Mpb of assembly that did not show any homology to known bacterial sequences. Identified communities in the two grains showed high concordance where Lactobacillus was the most abundant genus with a mapped abundance of 99.42% and 99.79%. This genus was dominantly represented by three species Lactobacillus kefiranofaciens, Lactobacillus buchneri and Lactobacillus helveticus with a total mapped abundance of 97.63% and 98.74%. We compared and verified our findings with 16S pyrosequencing and model based 16S data analysis. Our results suggest that microbial community profiling using whole genome shotgun data is feasible, can identify novel species data, and has the potential to generate a more accurate and detailed assessment of the underlying bacterial community, especially for low abundance species.

Genome-wide association analysis identifies new susceptibility loci for Behçet's disease and epistasis between HLA-B*51 and ERAP1.

Individuals with Behçet's disease suffer from episodic inflammation often affecting the orogenital mucosa, skin and eyes. To discover new susceptibility loci for Behçet's disease, we performed a genome-wide association study (GWAS) of 779,465 SNPs with imputed genotypes in 1,209 Turkish individuals with Behçet's disease and 1,278 controls. We identified new associations at CCR1, STAT4 and KLRC4. Additionally, two SNPs in ERAP1, encoding ERAP1 p.Asp575Asn and p.Arg725Gln alterations, recessively conferred disease risk. These findings were replicated in 1,468 independent Turkish and/or 1,352 Japanese samples (combined meta-analysis P < 2 × 10(-9)). We also found evidence for interaction between HLA-B*51 and ERAP1 (P = 9 × 10(-4)). The CCR1 and STAT4 variants were associated with gene expression differences. Three risk loci shared with ankylosing spondylitis and psoriasis (the MHC class I region, ERAP1 and IL23R and the MHC class I-ERAP1 interaction), as well as two loci shared with inflammatory bowel disease (IL23R and IL10) implicate shared pathogenic pathways in the spondyloarthritides and Behçet's disease.

A genome-wide analysis of lentivector integration sites using targeted sequence capture and next generation sequencing technology.

One application of next-generation sequencing (NGS) is the targeted resequencing of interested genes which has not been used in viral integration site analysis of gene therapy applications. Here, we combined targeted sequence capture array and next generation sequencing to address the whole genome profiling of viral integration sites. Human 293T and K562 cells were transduced with a HIV-1 derived vector. A custom made DNA probe sets targeted pLVTHM vector used to capture lentiviral vector/human genome junctions. The captured DNA was sequenced using GS FLX platform. Seven thousand four hundred and eighty four human genome sequences flanking the long terminal repeats (LTR) of pLVTHM fragment sequences matched with an identity of at least 98% and minimum 50 bp criteria in both cells. In total, 203 unique integration sites were identified. The integrations in both cell lines were totally distant from the CpG islands and from the transcription start sites and preferentially located in introns. A comparison between the two cell lines showed that the lentiviral-transduced DNA does not have the same preferred regions in the two different cell lines.