September,2015

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Molecular analysis of sarcoidosis lymph nodes for microorganisms: a case–control study with clinical correlates

The 16S gene fragment was amplified as previously described.8 The hsp65 gene was amplified using TB11 and TB12 primers, and the RNA polymerase subunit gene (rpoB) was amplified using MF and MR primers.9 The amplified products were then sequenced using the Big Dye Sequencing kit (Applied Biosystems, Foster City, California, USA) as per the vendor's recommended protocol. The sequences of two strands were assembled into double-stranded contig using Sequenchersoftware (Gene Codes, Ann Arbor, Michigan, USA).

Impact of HIV-1 Backbone on Neutralization Sensitivity: Neutralization Profiles of Heterologous Envelope Glycoproteins Expressed in Native Subtype C and CRF01_AE Backbone

DNA sequences were assembled and analyzed using Sequencher version 5.0 (Genecodes Inc., Ann Arbor, MI)

 

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S/MAR sequence confers long-term mitotic stability on non-integrating lentiviral vector episomes without selection

LV DNA was sequenced by Applied Biosystem’s 16-capillary 3130xl automated sequence analysis system by the OHSU sequencing core. The DNA fraction of the reaction mixture contains 6.4 pmol of primer (PPT-F, PBS-R) and 30 ng of purified DNA. Sequences were analyzed by Sequencherand BLAST software (NCBI).

 

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Monitoring Thiopurine Metabolites in Korean Pediatric Patients with Inflammatory Bowel Disease

All nine exons of the TPMT gene were amplified on a model 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA) using oligonucleotide primers and conditions as previously described with some modifications.20,21Direct sequencing was performed using ABI Big Dye Terminator kit v1.1 (Applied Biosystems) according to the manufacturer's instructions.

Human endogenous retroviral elements promote genome instability via non-allelic homologous recombination

The breakpoint region for each patient was determined by aligning Sanger sequencing reads to the HRG obtained from the UCSC Genome Browser using the Sequenchersoftware (Gene Codes Corporation, Ann Arbor, MI, USA). Breakpoint coordinates for each individual have been deposited in National Center for Biotechnology Information database of genomic structural variation (dbVar) and are available under the accession number [dbVar :nstd98].

 

Molecular Characterization of the First Ebola Virus Isolated in Italy, from a Health Care Worker Repatriated from Sierra Leone

We obtained the complete genome sequence of the virus isolate (Ebola virus/H. sapiens-tc/SLE/2014/Makona-Italy-INMI1) originating from a health worker evacuated from Sierra Leone to Italy in late November 2014. The virus was isolated on Vero E6 cells. Viral RNA was extracted (Roche) from the first passage; the complete genome was amplified in 45 overlapping fragments with EBOV-specific primers using the One-Step RT-PCR kit (Qiagen). The fragments were sequenced from both ends using Sanger techniques and assembled with the Sequencher software.

Independent Emergence of the Cosmopolitan Asian Chikungunya Virus, Phillipines 2012

All laboratory activities involving the virus isolates was conducted following BSL-2 biosafety practices and procedures in BSL-2 laboratory.  Viral RNA was extracted and screened for the CHIKV by using Reverse-transcription PCR (RT-PCR) previously described6. The E1 (N = 19) genes of positive sample were subsequently sequenced and analyzed to determine the virus genotype. Isolates identified as Asian genotype (N = 18) were subjected tofull genome sequencing using the Ion Torrent sequencing platform (Life Technologies, USA).

Complete Chloroplast Genome of Tanaecium tetragonolobum: The First Bignoniaceae Plastome

Contigs produced de novo were blasted against the original chloroplast genome reference in order to exclude contigs of nuclear origin. Contigs with coverage below 10x were eliminated, likely leading to the exclusion of contigs of mitochondrial origin as well. The remaining de novo and reference-guided contigs were assembled into larger contigs in Sequencher5.3.2 (Gene Codes Inc., Ann Arbor, MI) based on at least 20 bps overlap and 98% similarity.

Variability in phenotype induced by podocin variant R229Q plus a single pathogenic mutation

Genomic DNA was extracted from whole blood samples or saliva using standard methods. Mutation analysis was performed by direct sequencing of all eight exons of NPHS2, and the mutation bearing exons 8 and 9 of WT1 using exon-flanking primers. All mutations were confirmed with sequencing of the complementary strands. All sequences were analyzed using the Sequencher software package (Gene Codes Corp, Ann Arbor, MI). 

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