- Complete nontuberculous mycobacteria whole genomes using an optimized DNA extraction protocol for long-read. - Here we report a DNA extraction protocol that is optimized for long-read WGS of NTM, yielding large quantities of highly pure DNA with no additional clean-up steps.. - Results: Our DNA extraction method was compared to 6 other methods with variations in timing of mechanical disruption and enzymatic digestion of the cell wall, quantity of matrix material, and reagents used in extraction and precipitation. - avium subspecies hominissuis genomes using our extraction technique and the long-read sequencing MinION platform, including the identification of a novel plasmid.. - We expect that our finely-tuned extraction method will prove to be a valuable tool in long-read sequencing and completion of mycobacterial genomes going forward. - Utilization of comprehensive, long-read based approaches will advance the understanding evolution and pathogenicity of NTM infections.. - Keywords: Mycobacteria, Long-read sequencing, Whole genome sequencing, Nontuberculous mycobacteria, Mycobacterium avium complex, Mycobacterium abscessus complex, Genome assembly, Cystic fibrosis, Chronic obstructive pulmonary disease, Bronchiectasis. - 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Full list of author information is available at the end of the article. - Long-read sequencing promises an enhanced ability to complete bacterial genomes. - The most commonly avail- able techniques for long-read sequencing are the Single Molecule Real-Time (SMRT) technology by Pacific Bio- sciences® (PacBio, United States) and the newer Oxford Nanopore Technologies (ONT, United Kingdom) MinION . - Unlike most short-read sequencing methods, which require only very small amounts of DNA (as low as 1 ng), long-read platforms require high quantities of very pure DNA for acceptable processing. - DNA purity and integrity (i.e., length or molecular weight [MW]) is not only essential for functionality of the sequencer, but also is dir- ectly related to the quality of downstream bioinformatic analyses, as the DNA MW places a natural upper bound on the potential read length. - Standard ex- traction techniques (i.e., commercial kits) do not yield sufficient quantities of DNA for WGS while overly vigorous techniques shear DNA into suboptimal MWs for long-read sequencing. - The goal of developing our protocol was to extract large amounts of high MW, pure DNA for use in long- read WGS. - Our full DNA extraction protocol can be found in Additional file 1. - see Additional file 1 for TLB composition. - Method 5 is our optimized method and was the only method to produce sufficient quantity and quality standards for the ONT MinION long-read sequencer.. - Method 1 produced the highest total amounts of DNA (mean 12.45 μg, standard deviation [SD] 2.928). - Only Method 5 produced sufficient 260/230 for use with long- read sequencers, which was significantly higher than all other methods (Fig. - Notably, all 38 extractions using Method 5 yielded suffi- cient quality and quantity measurements for long-read se- quencing without requiring any additional clean-up steps (Fig. - All DNA extracts achieved high enough quan- tity of DNA (mean 4.17 μg, SD 0.80) and quality of DNA with mean 260/230 of 2.29 (SD 0.33) and mean 260/280 of 1.88 (SD 0.069), meeting the required specifications by Table 1 Differentiation of tested methods by variable. - a “ Early ” bead-beating refers to the timing prior to enzymatic digestion. - See Additional file 4: Figure S1. - 1 Quantity and quality of DNA by variable methods. - ONT whole genome sequencing and assembly. - Three isolates from MAC single colonies (CHOP101034, CHOP101115, and CHOP101174) were chosen for long- read sequencing as biological replicates. - As Method 5 was the only method to produce sufficient quantity and quality of DNA for long-read sequencing, use of an alternative method for comparison required additional clean-up steps (see Additional file 1 for isopropanol clean-up steps). - As clean-up of DNA inevitably results in significant loses of total DNA, we chose Method 1 for comparison, as it yielded the highest total DNA. - in the flow cell (Experiment protocol, SQK-RBK-004, ONT).. - Despite having sufficient quantity and quality of DNA and using the same library preparation and parameters for both run preparations, the Method 5 sequencing run gave superior total reads and total bases sequenced with significantly higher mean (p = 0.0168) and median (p = 0.0101) read lengths per barcode (Table 2). - Plots of sequencing run out- puts for all ONT MinION runs are displayed in Additional file 4: Figure S1. - The final, long-read based MAC genome assemblies were complete or near-complete with mean genome size 5.316 Mb and 69.01% GC content (Table 3). - Finished genome assemblies compared between two investigated methods of DNA extraction (Method 1 with isopropanol clean-up versus method 5 without clean-up) did not vary significantly by statistical analyses (paired t-tests) with regard to length, contig number, N50, %GC, or coverage. - 2 Quantity and quality of DNA extractions of 38 MAC and MABSC clinical isolates using optimized DNA extraction method. - While not reaching statistical significance, Method 5 genomes had lower overall contamination scores with means of 0.9 (Method 5, SD 0) versus 3.3 (Method 1, SD 2.12), p = 0.1210. - Method 5 genomes had significantly higher fine consistency with a mean of 97.37 (Method 5, SD 0.116) versus 96.2 (Method 1, SD 0.608), p = 0.0310.. - This could be reduced to as low as $80 per genome by including more barcodes in the sequencing run. - In summary, our optimized protocol for long-read DNA extraction and our assembly pipeline has allowed us to produce DNA sufficient for long-read sequencing after a single extraction without additional clean-up steps, and furthermore, allows us to present the first publicly-available M. - Two plasmids were identified in the assemblies, including a novel plasmid that we designate here as pMARIA (plasmid Mycobac- terium avium Replicon [class] 1 [type] a), and a plas- mid previously described by Caverly et al., pFLAC0181 (GenBank: CP023150.1, BioSample SAMN07528789, unpublished) identified by WGS from an isolate of M.. - CHOP101034 has 5,368,111 base pairs with 68.93% GC content, 5, 216 coding sequences (CDS), 99 repeat regions, 47 tRNAs, 3 rRNAs, and mean long-read ONT coverage of 83.12x. - Notably, Phred score was higher and error probability lower in the Method 5 run, which may reflect higher quality substrates. - b Completeness is the percentage of genes with universal roles represented in the genome. - Contamination approximates the percentage of the genome that is contaminated and is estimated by universal roles that are represented more than once in the genome [37]. - Our protocol produced DNA of sufficient quantity and quality for long-read whole genome sequencing with the ONT MinION sequencer. - Others have shown improved DNA purity with isopropanol extractions compared to cold ethanol ex- tractions with less salt carry-over, albeit at the expense of DNA yields [31, 32]. - The trademark of the mycobacterial cell wall is its hardy, heavily lipophilic exterior. - We reasoned that early mechanical disruption allows the exterior mycolic acid cell wall and peptidoglycan layer to be broken down first, with subse- quent enzymatic digestion with lysozyme and proteinase K to digest the remainder of the cell wall and expose its inner contents. - Although not achieving statistical significance in the head-to-head comparisons presented here, we have consistently noted increased shearing with late mechanical disruption, resulting in homogenously distributed smears. - The avoidance of clean-up steps is essential because repeat precipitation- based and SPRI bead-based clean-up methods consist- ently result in the loss of large amounts of DNA. - Thus, a single method that is able to produce highly pure DNA without clean-up is critical in cases where larger amounts of DNA are desired for long-read sequencing modalities. - However, even using the rapid kit with induced fragmentation of DNA and no size selection, we were able to complete bacterial genomes with our ONT-based assemblies.. - For example, CHOP101174 varies by 0.8% in length between the two sequencing methods and both methods produced closed genomes, however the measurements of consistency were higher and contamination lower in the Method 5 genome.. - In the setting of high GC content genomes with large repeat regions, a larger (and more fragmented) genome likely reflects duplications in repeat regions that cannot be resolved. - Long-read sequencing offers a much higher likelihood than short-read sequencing of producing complete (cir- cularized) genomes. - Our optimized extraction protocol and ONT assembly pipeline presented here were both sufficient and efficient for genome closure at a fraction of the cost and time of other approaches. - Undoubtedly, long-read assembled ge- nomes are the way of the future, but regardless of new technologies for cheap and high-fidelity DNA sequen- cing, we remain at the mercy of the cell wall, and we will continue to be faced with the delicate challenge of min- ing unscathed DNA from a distinctly robust substrate.. - We expect that our finely-tuned extraction method will prove to be a valuable tool in the mycobacterial genom- ics field going forward.. - DNA extraction method optimization and validation The following extraction protocol described is our opti- mized method, “Method 5.” Alterations in Method 5 for comparison are described in Table 1. - Method 3 is simi- lar to a standard protocol as described by Käser et al., with the only difference being the composition of the lysis buffer [32]. - DNA extraction method validation. - DNA was heated to 55–65 °C prior to quality assessment to ensure homogeneity of DNA per quality measurement guidelines [43]. - ONT whole genome sequencing and genome construction DNA preparation and sequencing. - Method 1 was chosen for comparative analyses to Method 5 as it had the highest total amount of DNA.. - Due to inadequate 260/230 of samples extracted by Method 1, these extracts required “clean-up”, which we completed by re-eluting in 100 μL of elution buffer and repeating isopropanol precipitation 2–3 times prior to achieving adequate quality measurements for use in the ONT MinION. - DNA from each of the 3 isolates was additionally sequenced by Illumina HiSeq 2500 using the Nextera XT library preparation kit (Illu- mina, US). - Genome assem- blies were constructed using the tool Unicycler (default settings, --mode normal, v0.4.8-beta) [48], which was cre- ated specifically for utilization of ONT long-reads in the assembly of bacterial genomes. - We additionally circularized with Circlator (circlator all, v using input of the Unicycler-assembled genome and ONT long-reads corrected by Canu (canu -correct, genomeSize = 5.2 m, errorRate = 0.144. - Coverage was calculated as an average of basecalled and trimmed fastq reads to the corresponding genome assembly using minimap2 alignment and taking the average read coverage of the samtools depth output (minimap2 -ax map-ont. - Quality and quantity measurements of DNA extractions were compared by one-way ANOVA with post-hoc Tukey’s multiple comparison tests and un- paired t-tests when appropriate. - Quality measurements of the genomes were additionally con- ducted by analyzing presence/absence and frequency of universal genes represented in the genomes in PATRIC, which were compared by unpaired t-tests [54].. - Additional file 1. - Protocol for DNA Extraction of Nontuberculous Mycobacteria for Long-Read Whole Genome Sequencing.. - Additional file 2: Table S1. - Additional file 3: Table S2. - There were no significant differences between MAC and MAB strains when comparing any of the listed statistics (unpaired, parametric t-test), nor were there any significant differences in any listed statistics when compared to the previous ONT sequencing run without size selection. - Additional file 4: Figure S1. - Additional file 5: Table S4. - Additional file 6: Table S3. - NTM: Nontuberculous mycobacteria;. - SEM: Standard error of the mean. - WGS: Whole genome sequencing. - JMB and PJP have substantially contributed to the drafting and revision of the work and have approved the submitted version. - JMB and PJP agree to be personally accountable for their own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.. - Please see “ Data Accession ” in the Results section for identification of reads and genomes associated with specific NCBI BioSamples.. - The research presented here was approved by the Children ’ s Hospital of Philadelphia Institutional Review Board (CHOP IRB for the retrospective collection and sequencing of microbiologic specimens stored in the CHOP Clinical Microbiology Laboratory. - Current epidemiologic trends of the Nontuberculous mycobacteria (NTM). - Epidemiology of nontuberculous mycobacteria (NTM) amongst individuals with cystic fibrosis (CF). - Genome sequencing of Mycobacterium abscessus isolates from patients in the United States and comparisons to globally diverse clinical strains. - during chronic infection of the human lung. - Functional characterization of the Mycobacterium abscessus genome coupled with condition specific transcriptomics reveals conserved molecular strategies for host adaptation and persistence. - Phylogenomics and comparative genomic studies robustly support division of the genus Mycobacterium into an emended genus Mycobacterium and four novel genera. - genome sequencing by long-read sequencer using SMRT technology in medical area. - 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