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Protocol publication

[…] ct sequencing., To prepare a next-generation sequencing library, genomic DNA was extracted from PBMCs, and randomly fragmented by a Covaris Adaptive Focused Acoustics™ (AFA) instrument to obtain an average fragment size of 200 to 300 base pairs. Then, the genomic DNA fragments were end-repaired, adenylated, linked to Illumina-specific adapter sequences, and amplified by PCR. Exome capture was performed using the TruSeq™ Exome Enrichment Kit (Illumina, San Diego, CA, USA). Sequencing was performed on the Illumina Genome Analyzer HiSeq2000 following the manufacturer’s instructions. The average coverage was 20-fold. The sequencing reads were aligned to the human reference genome hg19 using the Burrows-Wheeler Aligner (BWA) software. We analyzed the data using the Genome Analysis Toolkit (GATK) variant pipeline with respect to 1,000 Genomes Data, Single Nucleotide Polymorphism Database (dbSNP) and in-house data, and we used the SnpEff software to predict the effects of sequence variants., CSF and/or serum of the patient, patient family members and unrelated controls were used for laboratory analyses on the same day of the sampling or were stored at −40°C in the serum and CSF bank of the Neurological Institute ‘Carlo Besta’. Control CSF was obtained from patients with non-inflammatory neurological diseases (that is, brain tumors, neurodegenerative and heredo-metabolic diseases)., IL-1β and soluble IL-6R (sIL-6R) were determined by ELISAs (Human IL-1β/IL-1F2 Quantikine ELISA Kit, catalogue number DLB50, and Human IL-6 R alpha Quantikine ELISA Kit, catalogue number DR600; R&D Systems, Minneapolis, MN, USA; http:// […]

Pipeline specifications

Software tools BWA, GATK, SnpEff
Organisms Homo sapiens
Diseases Central Nervous System Diseases, Disease Attributes, Inflammation, Meningitis, Nervous System Diseases, Hearing Disorders, Sensation Disorders, Neurologic Manifestations, Hereditary Autoinflammatory Diseases, Brain Diseases