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SRX1621511: RNA-seq of PT-5 brain biopsy (occipital). Sequencing diagnosis: JC polyomavirus
1 ILLUMINA (Illumina MiSeq) run: 25,050 spots, 3.4M bases, 1.8Mb downloads

Design: Tissue for the study was collected under a research protocol approved by the Johns Hopkins University School of Medicine Institutional Review Board (IRB NA_00003551). Fresh frozen tissues were obtained from eight cases and two were from paraffin-processed tissues. Except for the paraffin-processed tissues, the small amounts of biopsy material were consumed completely by the library preparation process, leaving no material for additional studies. Fresh frozen tissues used for RNA isolation were submerged immediately after biopsy in RNALater. DNA or RNA isolation, library preparation, and sequencing on Illumina MiSeq platform were all performed in the Johns Hopkins Deep Sequencing and Microarray Core Facility. All samples were first treated with lyticase to break down fungal cell walls before proceeding to either DNA or RNA isolation to ensure capture of any potential fungal sequences in the sample. Biopsy tissue was snap frozen for DNA isolation or preserved in RNALater for RNA isolation. Both type of tissue were homogenized in 180 ul Y1 (1M sorbitol with 0.1M EDTA and b-mercaptoethanol) solution and treated with 20ul 1U/ul lyticase at 30°C for 30 minutes with rotation. DNA was then isolated following the QiaAmp DNA mini-protocol, and RNA was isolated following the Qiagen miRNeasy protocol for total RNA. Library preparation was performed using either Illumina DNA Nano sample preparation kit or Illumina TruSeq stranded total RNA kit for DNA samples (depending on the available quantity of DNA) and Nugen RNAseq library kit for RNA samples. For each sample, one run (no multiplexing) of an Illumina MiSeq instrument was used for sequencing.
Submitted by: Johns Hopkins School of Medicine
Study: Metagenomic sequencing for neuropathological diagnosis of infections of the nervous system
show Abstracthide Abstract
In a prospective-pilot study, we applied NGS in combination with a new computational analysis pipeline to detect the presence of pathogenic microbes in brain or spinal cord biopsies from ten patients with neurological problems indicating possible infection but for whom conventional clinical and microbiology studies yielded negative or inconclusive results. Direct DNA and RNA sequencing of brain tissue biopsies successfully identified with high confidence the infectious agent in three patients, identified possible pathogens in two more, and helped to understand neuropathological processes in three others, demonstrating the power of large-scale unbiased sequencing as a novel diagnostic tool.
Sample: PT-5
SAMN04529566 • SRS1330176 • All experiments • All runs
Organism: JC polyomavirus
Library:
Name: PT-5
Instrument: Illumina MiSeq
Strategy: RNA-Seq
Source: METAGENOMIC
Selection: RANDOM PCR
Layout: SINGLE
Runs: 1 run, 25,050 spots, 3.4M bases, 1.8Mb
Run# of Spots# of BasesSizePublished
SRR321409225,0503.4M1.8Mb2016-03-14

ID:
2314000

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