Pulmonary Metagenomic Sequencing Suggests Missed Infections in Immunocompromised Children

Zinter, Matt S.; Dvorak, Christopher C.; Mayday, Madeline Y; Iwanaga, Kensho; Ly, Ngoc P.; McGarry, Meghan E.; Church, Gwynne; Faricy, Lauren Elizabeth; Rowan, Courtney M.; Hume, Janet R.; Steiner, Marie; Crawford, Emily; Langelier, Charles; Kalantar, Katrina; Chow, Eric D.; Miller, Steve; Shimano, Kristen; Melton, Alexis; Yanik, Gregory A.; Sapru, Anil; DeRisi, Joseph L.

doi:10.1093/cid/ciy802

Cited by 120 publications

(73 citation statements)

References 62 publications

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“…Early studies of nasopharyngeal aspirates from lower respiratory tract infections in China (10) and Sweden (11) revealed numerous viruses, with members of the families Paramyxoviridae (RSV, metapneumovirus, and parainfluenza virus), Picornaviridae (rhinovirus), and Orthomyxoviridae (influenza virus) predominating. Numerous other metagenomic studies of clinical respiratory samples have confirmed the ability of this method to detect diverse human viruses (12)(13)(14)(15)(16)(17). Metagenomic analyses thus show great promise as a supplement to or even replacement for more specific viral genome detection assays, although sensitivity issues remain (18)(19)(20)(21).…”

Section: Discussionmentioning

confidence: 93%

Effect of Geographic Isolation on the Nasal Virome of Indigenous Children

Altan

Dib

Gulloso³

et al. 2019

J Virol

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The influence of living in small remote villages on the diversity of viruses in the nasal mucosa was investigated in three Colombian villages with very different levels of geographic isolation. Metagenomic analysis was used to characterize viral nucleic acids in nasal swabs from 63 apparently healthy young children. Sequences from human virus members of the families Anelloviridae, Papillomaviridae, Picornaviridae, Herpesviridae, Polyomaviridae, Adenoviridae, and Paramyxoviridae were detected in decreasing proportions of children. The number of papillomavirus infections detected was greater among Hispanic children most exposed to outside contacts, while anellovirus infections were more common in the isolated indigenous villages. The diversity of the other human viruses detected did not differ among the villages. Closely related variants of rhinovirus A or B were identified in 2 to 4 children from each village, reflecting ongoing transmission clusters. Genomes of viruses not currently known to infect humans, including members of the families Parvoviridae, Partitiviridae, Dicistroviridae, and Iflaviridae and circular Rep-encoding single-stranded DNA (CRESS-DNA) virus, were also detected in nasal swabs, possibly reflecting environmental contamination from insect, fungal, or unknown sources. Despite the high levels of geographic and cultural isolation, the overall diversity of human viruses in the nasal passages of children was not reduced in highly isolated indigenous villages, indicating ongoing exposure to globally circulating viruses. IMPORTANCE Extreme geographic and cultural isolation can still be found in some indigenous South American villages. Such isolation may be expected to limit the introduction of otherwise common and widely distributed viruses. Very small population sizes may also result in rapid local viral extinction due to a lack of seronegative subjects to maintain transmission chains for rapidly cleared viruses. We compared the viruses in the nasal passages of young children in three villages with increasing levels of geographic isolation. We found that isolation did not reduce the overall diversity of viral infections. Multiple infections with nearly identical rhinoviruses could be detected within each village, likely reflecting recent viral introductions and transmission clusters among epidemiologically linked members of these very small communities. We conclude that, despite their geographic isolation, remote indigenous villages show evidence of ongoing exposure to globally circulating viruses.

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Section: Discussionmentioning

confidence: 93%

Effect of Geographic Isolation on the Nasal Virome of Indigenous Children

Altan

Dib

Gulloso³

et al. 2019

J Virol

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“…Our laboratory has previously described a mNGS library prep protocol for RNA using the New England Biolabs Ultra II Library Prep Kit [13–15]. In this protocol, RNA was quantified using QuBit, fragmented in a magnesium-based buffer at 94°C, primed with random hexamers, and reverse transcribed to form cDNA.…”

Section: Methods and Resultsmentioning

confidence: 99%

“…Final HeLa RNA libraries were sequenced on the Illumina MiSeq to an average depth of 2.5 million paired-end reads. Resultant data were processed through a pipeline for pathogen detection developed in our laboratory involving subsequent removal of duplicate reads, reads with low quality, and reads aligning to phage [13,16,17]. Original FASTQ files are available at BioProject Accession #PRJNA493096.…”

Section: Methods and Resultsmentioning

confidence: 99%

Miniaturization and optimization of 384-well compatible metagenomic sequencing library preparation

et al. 2018

Preprint

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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...

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“…Given the sensitivity of mNGS, it is common to identify contaminating microbial sequences derived from laboratory contaminants, reagents, collection tubes, etc. There exist numerous approaches to assist in distinguishing background contaminants from true microbes [34,53,61]. IDseq implements a previously described z-score method for background correction [53].…”

Section: Idseq Z-score and Aggregate Score Metricsmentioning

confidence: 99%

IDseq – An Open Source Cloud-based Pipeline and Analysis Service for Metagenomic Pathogen Detection and Monitoring

Kalantar

Carvalho

Bourcy

et al. 2020

Preprint

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Background: Metagenomic next generation sequencing (mNGS) has enabled the rapid, unbiased detection and identification of microbes without pathogen-specific reagents, culturing, or a priori knowledge of the microbial landscape. mNGS data analysis requires a series of computationally intensive processing steps to accurately determine the microbial composition of a sample. Existing mNGS data analysis tools typically require bioinformatics expertise and access to local server-class hardware resources. For many research laboratories, this presents an obstacle, especially in resource limited environments. Findings: We present IDseq, an open source cloud-based metagenomics pipeline and service for global pathogen detection and monitoring (https://idseq.net). The IDseq Portal accepts raw mNGS data, performs host and quality filtration steps, then executes an assembly-based alignment pipeline which results in the assignment of reads and contigs to taxonomic categories. The taxonomic relative abundances are reported and visualized in an easy-to-use web application to facilitate data interpretation and hypothesis generation. Furthermore, IDseq supports environmental background model generation and automatic internal spike-in control recognition, providing statistics which are critical for data interpretation. IDseq was designed with the specific intent of detecting novel pathogens. Here, we benchmark novel virus detection capability using both synthetically evolved viral sequences, and real-world samples, including IDseq analysis of a nasopharyngeal swab sample acquired and processed locally in Cambodia from a tourist from Wuhan, China, infected with the recently emergent SARS-CoV-2. Conclusion: The IDseq Portal reduces the barrier to entry for mNGS data analysis and enables bench scientists, clinicians, and bioinformaticians to gain insight from mNGS datasets for both known and novel pathogens.

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Pulmonary Metagenomic Sequencing Suggests Missed Infections in Immunocompromised Children

Cited by 120 publications

References 62 publications

Effect of Geographic Isolation on the Nasal Virome of Indigenous Children

Effect of Geographic Isolation on the Nasal Virome of Indigenous Children

Miniaturization and optimization of 384-well compatible metagenomic sequencing library preparation

IDseq – An Open Source Cloud-based Pipeline and Analysis Service for Metagenomic Pathogen Detection and Monitoring

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