Question: How can metagenomic next-generation sequencing of cerebrospinal fluid be leveraged to aid in the diagnosis of patients with subacute or chronic meningitis?Findings: Metagenomic next-generation sequencing identified parasitic worms, fungi and viruses in a case series of seven subjects. A database of water-only and healthy patient controls enabled application of a z-score based scoring algorithm to effectively separate bona fide pathogen sequences from spurious environmental sequences. Meaning:Our scoring algorithm greatly simplified data interpretation in a series of patients with a wide range of challenging infectious causes of subacute or chronic meningitis identified by metagenomic next-generation sequencing. AbstractImportance: Identifying infectious causes of subacute and chronic meningitis can be challenging. Enhanced, unbiased diagnostic approaches are needed.Objective: To present a case series of patients with diagnostically challenging subacute and chronic meningitis in whom metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF), supported by a statistical framework generated from mNGS sequencing of non-infectious patients and environmental controls, identified a pathogen.Design: Case series. Using mNGS data from the CSF of 94 non-infectious neuroinflammatory cases and 24 water and reagent controls, we developed and implemented a weighted scoring metric based on z-scores at the species and genus level for both nucleotide and protein databases to prioritize and rank mNGS results. We . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/213561 doi: bioRxiv preprint first posted online Nov. 7, 2017; performed mNGS on total RNA extracted from CSF of patients with subacute or chronic meningitis and highlight seven cases representing a diverse array of pathogens.Setting: A multi-center study of mNGS pathogen discovery in patients with suspected neuroinflammatory conditions. Participants:Patients with diagnostically challenging subacute or chronic meningitis enrolled in a research study of mNGS performed on CSF.Intervention: mNGS was performed on total RNA extracted from CSF (0.25-0.5 mL). A weighted z-score was used to filter out environmental contaminants and facilitate efficient data triage and analysis.Main Outcomes: 1) Pathogens identified by mNGS and 2) ability of a statistical model to prioritize, rank, and simplify mNGS results.
17 Preparation of high-quality sequencing libraries is a costly and time-consuming component of 18 metagenomic next generation sequencing (mNGS). While the overall cost of sequencing has 19 dropped significantly over recent years, the reagents needed to prepare sequencing samples are 20 likely to become the dominant expense in the process. Furthermore, libraries prepared by hand 21 are subject to human variability and needless waste due to limitations of manual pipetting 22 volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of 23 reagents without consumable pipette tips, has the potential to provide significant advantages.24 Here, we describe the integration of several instruments, including the Labcyte Echo 525 25 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize 26 and automate mNGS library preparation, significantly reducing the cost and the time required to 27 prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in 28 RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full 29 volume reactions. Furthermore, detection of viral and microbial species from cell culture and 30 patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library 31 preparations, we achieved a savings of over 80% in materials and reagents alone, and reduced 32 preparation time by 90% compared to manual approaches, without compromising quality or 33 representation within the library. 34 35 3 36 Introduction 37 Metagenomic next-generation sequencing (mNGS) is becoming an increasingly useful tool in the 38 field of biology and clinical medicine. Its applications are almost limitless -any nucleic acid 39 can be turned into a library, amplified, and sequenced, making mNGS an appealing technology 40 for labs and hospitals alike. As sequencers such as the Illumina NovaSeq increase throughput, 41 hundreds to thousands of libraries can be sequenced in a single run. Although the per-base cost 42 of sequencing has become less expensive over the last several decades, the cost and time 43 associated with sample preparation remain disproportionately high [1,2]. 44 45 Manual library preparation is tedious and is often the bottleneck for many sequencing projects. 46 Numerous library preparation protocols have been adapted for automation through the use of 47 various positive displacement tip-based liquid handler instruments, including the Beckman 48 Coulter Biomek, Hamilton Star, Agilent Technologies Bravo, TTP LabTech Mosquito, and 49 others [3-5]. Though these provide more hands-off time during the library preparation process, 50 the overall cost can often exceed that of hand-prepared libraries due to the increased dead 51 volume of reagents and the large number of expensive, sometimes proprietary tips required for 52 liquid handlers. Furthermore, sub-microliter miniaturization is a challenge for the majority of 53 positive displacement based liquid handlers. 54 55 Recently, ac...
ROHHAD (Rapid-onset Obesity with Hypothalamic Dysfunction, Hypoventilation and Autonomic Dysregulation) is a rare, yet severe pediatric disorder resulting in hypothalamic dysfunction and frequent sudden death. Genetic and other investigations have failed to identify an etiology or diagnostic test. Frequent co-occurrence of neuroblastic tumors (NTs) and cerebrospinal fluid inflammation point to an autoimmune paraneoplastic neurological syndrome (PNS); however, specific anti-neural autoantibodies, a hallmark of PNS, have not been identified. Here, we screened antibodies from a curated cohort of ROHHAD patients (n=9) and controls (n=150) using a programmable phage display of the human peptidome (PhIP-Seq). Our ROHHAD cohort exhibited frequent association with NTs (8/9) and features consistent with autoimmune etiology. Autoantibodies to Zinc finger and SCAN domain-containing protein 1 (ZSCAN1) were discovered and orthogonally validated in 7 of 9 ROHHAD patients, all of whom had NTs, and shown to be absent in non-ROHHAD pediatric patients with NTs. Notably, human ZSCAN1 expression was confirmed in ROHHAD tumor and healthy human hypothalamus. Our results support the notion that tumor-associated ROHHAD is a pediatric PNS, potentially initiated by an immune response to peripheral NT. ZSCAN1 autoantibodies may aid in an accurate diagnosis of ROHHAD, thus providing a means toward early detection and treatment. Lastly, given the absence of the ZSCAN1 gene in rodents, our study highlights the value of human-based approaches in addition to the classical rodent-based approaches for detecting novel PNS subtypes.
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