DOI: 10.5061/dryad.2ngf1vj25
title: Evaluating the diagnostic capabilities of nanopore sequencing for <em>Borrelia burgdorferi</em> detection in blacklegged ticks

description: Nanopore adaptive sampling was performed across seven sequencing
 experiments on 168 wild-caught adult blacklegged ticks. Nanopore adaptive
 sampling was leveraged to enrich for all known bacterial pathogens
 vectored by blacklegged ticks in the US. Raw POD5 files from the
 sequencing experiments were re-basecalled using the super high-accuracy
 basecalling model in MinKNOW. The resulting dataset provided here contains
 the concatenated zipped FASTQ files for the rebasecalled sequence data,
 with each file representing an individual tick. Unique identifiers
 correspond to the locations where ticks were collected in Minnesota.
description: <strong>Sample selection</strong>
 Following PCR analysis, we randomly selected total gDNA extracts
 from 70% *B. burgdorferi-*positive and 30% <em>B.
 burgdorferi</em>-negative ticks for nanopore sequencing experiments.
 Briefly, positive and negative samples were separated into two datasets,
 and a random subsample was drawn from each. An infection prevalence of 70%
 and a likely sensitivity of 0.80 (null hypothesis) to 0.90 (alternative
 hypothesis) requires a total of 153 samples (power: 0.819; p-value:
 0.040). Each library preparation kit has the capability to multiplex 24
 samples and to ensure a sufficient initial library concentration (&gt;
 1 ug), we increased the sample size to 168 total gDNA extracts for
 sequencing. Each library contained the same proportion (i.e., 70:30) of
 nPCR-positive and nPCR-negative samples, respectively. To ensure blinding,
 ticks from negative and positive nPCR-infected classes were selected for a
 specific run using a random number generator to determine their inclusion
 and relative barcode order. Samples from negative and positive infected
 classes were prioritized according to their DNA concentration and quality
 to maximize the input library concentration for downstream sequencing. All
 subsequent analyses using sequence data were performed blinded to
 infection status. ONT native barcoding recommends that each barcode have
 an initial DNA concentration of 400 ng, and gDNA extracts that did not
 meet this threshold were subject to vacuum centrifuge concentration using
 a Vacufuge Plus (Eppendorf). Concentrated gDNA extracts were quantified
 using a NanoDrop One fluorometer before library preparation. DNA inputs
 were not normalized before library preparation.
 <strong>Library preparation</strong>
 Seven individual libraries, each consisting of 24 ticks, were
 constructed using the ONT Native Barcoding Kit (SQK-NBD114) following the
 manufacturer’s instructions for use with a MinION flow cell (R10.4.1). All
 libraries were prepared identically. DNA ends were initially repaired and
 prepared for barcode and adapter ligation using the NEBNext FFPE DNA
 Repair Mix and NEBNext Ultra II End Repair/dA-tailing Module (New England
 Biolabs Inc., Ipswich, MA). Each sample was incubated at 20 °C and 65 °C
 for 5 minutes, respectively, before purification and concentration using
 AMPure XP magnetic beads (Beckman Coulter, A63881). A 1:1X ratio of
 magnetic beads was used at each bead cleanup step. The samples were then
 cleaned with 80% EtOH and eluted in 10 μL of nuclease-free water. ONT
 molecular barcodes (Oxford Nanopore Technologies) were added to the
 eluate, enabling pooling, using a Blunt/TA Ligase Master Mix (New England
 Biolabs Inc.), and incubated for 20 minutes at room temperature. Following
 incubation, 4 μL of EDTA (Oxford Nanopore Technologies), which acts as a
 chelating agent to inhibit exonucleases from degrading the DNA, was added
 to each sample, and all samples were pooled into a single tube. The
 pooled, barcoded library was then purified and concentrated using AMPure
 XP magnetic beads, incubated at room temperature, cleaned using 80% EtOH,
 and eluted in 35 μL of nuclease-free water. Native adapters (ONT) were
 then added to the pooled barcoded library, which serve to anchor DNA
 fragments to individual nanopores on the flow cell for sequencing, using
 the NEBNext Quick Ligation Reaction Buffer and Quick T4 DNA ligase (New
 England Biolabs Inc.), and incubated for 20 minutes at room temperature.
 The library was then cleaned and purified using AMPure XP magnetic beads,
 incubated at room temperature, and cleaned using the ONT short fragment
 buffer. The final pooled library was eluted in 15 μL of Elution Buffer
 (ONT). At each elution step, the libraries were quantified using a Qubit 4
 Fluorometer with the dsDNA HS Assay Kit (Thermo Fischer Scientific,
 Q32854). <strong>Sequencing and
 basecalling</strong> Each barcoded library (n=7)
 was sequenced using individual MinION Flow Cells (R10.4.1) on a MinION
 Mk1B instrument (ONT). Sequencing experiments were performed on a desktop
 computer using Pop!_OS with the following specifications: AMD Ryzen 9
 7900X 12-core processor (Advanced Micro Devices, Inc., Santa Clara, United
 States), NVIDIA GeForce RTX 4090 (Nvidia, Santa Clara, United States), 64
 GB RAM, 4 TB SSD, and an 8 TB internal hard drive. Sequence experiments
 were parameterized and monitored using MinKNOW (ONT). Each flow cell was
 checked to ensure that an adequate (i.e., &gt;800) number of pores
 were available for sequencing before loading each of the final libraries.
 Flow cells were primed with a mixture of ONT Flow Cell Flush and Flow Cell
 Tether (ONT) that was gently loaded into the priming port, avoiding any
 introduction of air bubbles. Each library was prepared with ONT Sequencing
 Buffer and Library Beads (ONT) and then loaded onto the flow cell's
 SpotOn sample port. After loading, sequencing parameters were specified
 using MinKNOW, specifically enabling the enrichment of specific sequences
 as they pass through individual nanopores, termed nanopore adaptive
 sampling (NAS). NAS allows users to selectively enrich or deplete for
 target sequences, including whole genomes. As a DNA strand passes through
 the nanopore, the electrical signal is disrupted, producing a distinct
 change that represents the passing nucleotide sequence, which is
 basecalled in real-time using Dorado v0.8.1 (ONT). Nanopore adaptive
 sampling takes the first few hundred bases of the passing nucleotide
 sequence and maps it to the user-specified reference file. When enrichment
 is selected, if the passing nucleotide sequence is 70% similar to any
 sequence in the reference file, the sequencing of that DNA fragment will
 continue to completion. However, if the sequenced fragment is &lt;70%
 similar, it is ejected from the nanopore, and the pore is freed to
 sequence a new strand. Here, each sequencing experiment was performed with
 an enrichment file containing known DNA-based TBPs vectored by the
 blacklegged tick: <em>Borrelia burgdorferi sensu stricto</em>
 (GCF_000008685), <em>Borrelia miyamotoi</em> (GCF_019668505),
 <em>Ehrlichia muris euclairensis</em> (GCF_000508225),
 <em>Babesia microti</em> (GCF_000691945), and
 <em>Anaplasma phagocytophilum</em> (GCF_000439775). Each
 reference genome FASTA file was downloaded from NCBI, concatenated using
 the command line into a penultimate reference file, and indexed using
 minimap2 [21]. The indexed pathogen reference file was selected for enrichment and alignment in MinKNOW before starting each sequencing experiment. Additional sequencing parameters were specified in MinKNOW before sequencing, including read quality (Q&gt;8), minimum read length (&gt;250 bp), the location to write output files, and library preparation kits used to construct libraries. Each flow cell was sequenced to exhaustion (e.g., 72 hours) without washing and reloading individual libraries. <strong>Basecalling and pre-processing</strong> Raw data from each sequencing experiment in POD5 file format were rebasecalled, adapter trimmed, and demultiplexed using Dorado with the “super accuracy” model dna_r10.4.1_e8.2_400bps_supv4.2.0. Passed FASTQ files, which included reads with a quality score greater than 8 and a minimum read length of 250 bp, were extracted and utilized for downstream analysis. Each barcode (n=24 in each of 7 experiments) contained multiple zipped FASTQ files for every sequenced DNA fragment that were concatenated using the command line into single zipped FASTQ files comprising all reads for a given barcode.
description: # Evaluating the diagnostic capabilities of nanopore sequencing for
 *Borrelia burgdorferi* detection in blacklegged ticks Dataset DOI:
 [10.5061/dryad.2ngf1vj25](10.5061/dryad.2ngf1vj25) ## Description of the
 data and file structure Blacklegged ticks were collected from three sites
 across Minnesota, including Washington County, Anoka County, and Winona
 County. 275 adult blacklegged ticks were collected and analyzed by nPCR to
 detect the presence of *Borrelia burgdorferi*. Ticks were morphologically
 validated before molecular analysis. Positive and negative (control) ticks
 were randomly selected for sequencing to determine the diagnostic
 capabilities of nanopore adaptive sampling as a rapid molecular
 surveillance tool. A total of 168 tick genomic DNA extracts were subject
 to nanopore sequencing. Seven sequencing experiments (n=24 each) were
 conducted with nanopore adaptive sampling targeting the whole genome of
 *B. burgdorferi*. Each sequencing experiment was performed on individual
 flow cells using a MinION instrument. This dataset includes the super high
 accuracy basecalled nanopore sequencing reads as zipped FASTQ files
 (n=168) for each tick within a zipped tarball file. ### Files and
 variables #### File: all_fastqs.tar.gz **Description:** Zipped tarball
 file containing zipped FASTQ files for each sequenced blacklegged tick
 (n=168). The respective zipped FASTQ files contain nanopore adaptive
 sampling FASTQs that were concatenated for each individual. Data was
 rebasecalled using the super high accuracy basecalling model in MinKNOW.
 ## Code/software To extract the tarball file and obtain the zipped FASTQ
 files for each adult blacklegged tick that was sequenced: tar -xzvf
 all_fastqs.tar.gz #