When SNPs are not enough: Molecular epidemiology of a ...
MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island Philip M Ashtona^, Satheesh Naira^, Tim Dallmana*, Salvatore Rubinob,c, Wolfgang Rabschd, Solomon Mwaigwisyae, John Waine, and Justin O’Gradye*.a Gastrointestinal Bacteria Reference Unit, Public Health England, Colindale, London, UK.b Department of Biomedical Sciences, University of Sassari, Sassari, Italy.c Department of Infection and Immunity, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabiad National Reference Centre for Salmonellae and other Enterics, Robert Koch Institute, Wernigerode, Germany. e Norwich Medical School, University of East Anglia, Norwich, UK.^ Authors contributed equallyCorrespondence should be addressed to J.O’G (justin.ogrady@uea.ac.uk) or T.D. (tim.dallman@.uk).Short-read, high throughput sequencing technology cannot identify the chromosomal position of repetitive insertion sequences that typically flank horizontally acquired genes such as bacterial virulence genes and antibiotic resistance genes. The MinION? nanopore sequencer can produce long sequencing reads on a device similar in size to a USB memory stick. Here we apply a MinION? sequencer to resolve the structure and chromosomal insertion site of a composite antibiotic resistance island in Salmonella Typhi Haplotype 58. Nanopore sequencing data from a single 18-hour run was used to create a scaffold on which to assemble short-read Illumina data. Our results demonstrate the potential of the MinION device in clinical laboratories to track the epidemic spread of bacterial pathogensShort read, high throughput next generation sequencing (NGS) technology has tranformed our understanding of microbiology and is poised to become an integral tool in epidemiologyPEVuZE5vdGU+PENpdGU+PEF1dGhvcj5VbmRlcndvb2Q8L0F1dGhvcj48WWVhcj4yMDEzPC9ZZWFy
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ADDIN EN.CITE.DATA 1. Although the utility of whole genome sequencing (WGS) for public health infection control is clear, adoption in clinical microbiology laboratories has been limited ADDIN EN.CITE <EndNote><Cite><Author>Wain</Author><Year>2013</Year><RecNum>31</RecNum><DisplayText><style face="superscript">2</style></DisplayText><record><rec-number>31</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">31</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Wain, J.</author><author>Mavrogiorgou, E.</author></authors></contributors><titles><title>Next-generation sequencing in clinical microbiology</title><secondary-title>Expert Rev Mol Diagn</secondary-title><alt-title>Expert review of molecular diagnostics</alt-title></titles><pages>225-7</pages><volume>13</volume><number>3</number><keywords><keyword>Bacterial Infections/diagnosis/microbiology</keyword><keyword>*High-Throughput Nucleotide Sequencing</keyword><keyword>Humans</keyword><keyword>Microbiology</keyword><keyword>*Molecular Diagnostic Techniques</keyword><keyword>*Molecular Typing</keyword><keyword>Mycoses/diagnosis/microbiology</keyword><keyword>Parasitic Diseases/diagnosis/parasitology</keyword><keyword>Sequence Analysis, DNA</keyword><keyword>Virus Diseases/diagnosis/virology</keyword></keywords><dates><year>2013</year><pub-dates><date>Apr</date></pub-dates></dates><isbn>1744-8352 (Electronic)
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ADDIN EN.CITE.DATA 3 due to their inherent repetitive nature or the flanking of these elements by repetitive insertion sequences. Analysing these regions is essential for determining key characteristics such as antibiotic resistance profiles and for identifying highly pathogenic variants of many bacterial species ADDIN EN.CITE <EndNote><Cite><Author>Livermore</Author><Year>2013</Year><RecNum>4</RecNum><DisplayText><style face="superscript">4</style></DisplayText><record><rec-number>4</rec-number><foreign-keys><key app="EN" db-id="etv0xzxs0rvef0ef5z95xf0pzrfzs0x5rva9">4</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Livermore, D. M.</author><author>Wain, J.</author></authors></contributors><auth-address>Norwich Medical School, University of East Anglia, Norwich, Norfolk, NR4 7TJ, United Kingdom.</auth-address><titles><title>Revolutionising bacteriology to improve treatment outcomes and antibiotic stewardship</title><secondary-title>Infect Chemother</secondary-title><alt-title>Infection & chemotherapy</alt-title></titles><periodical><full-title>Infect Chemother</full-title><abbr-1>Infection & chemotherapy</abbr-1></periodical><alt-periodical><full-title>Infect Chemother</full-title><abbr-1>Infection & chemotherapy</abbr-1></alt-periodical><pages>1-10</pages><volume>45</volume><number>1</number><dates><year>2013</year><pub-dates><date>Mar</date></pub-dates></dates><isbn>2093-2340 (Print)
1598-8112 (Linking)</isbn><accession-num>24265945</accession-num><urls><related-urls><url>. Currently gap closure requires extensive post-sequencing laboratory based analysis, which can take several months and makes the results irrelevant for clinical diagnostics and for guiding public health interventions.Sequencing technology that generates long reads, capable of spanning repetitive sequences and closing gaps in short read data, is commercially available (Pacific Biosystems PacBio RS II) but has significant capital cost outlay, a very large laboratory footprint and is technically demanding. DNA sequencing using nanopore technology is an alternative method for producing long read sequence data but has been a specialised research tool until very recently ADDIN EN.CITE <EndNote><Cite><Author>Clarke</Author><Year>2009</Year><RecNum>38</RecNum><DisplayText><style face="superscript">5</style></DisplayText><record><rec-number>38</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">38</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Clarke, J.</author><author>Wu, H. C.</author><author>Jayasinghe, L.</author><author>Patel, A.</author><author>Reid, S.</author><author>Bayley, H.</author></authors></contributors><auth-address>Oxford Nanopore Technologies Ltd, Begbroke Science Park, Sandy Lane, Oxford OX5 1PF, UK.</auth-address><titles><title>Continuous base identification for single-molecule nanopore DNA sequencing</title><secondary-title>Nat Nanotechnol</secondary-title><alt-title>Nature nanotechnology</alt-title></titles><pages>265-70</pages><volume>4</volume><number>4</number><keywords><keyword>Base Sequence</keyword><keyword>Crystallization/*methods</keyword><keyword>DNA/*analysis/*chemistry</keyword><keyword>Macromolecular Substances/chemistry</keyword><keyword>Materials Testing</keyword><keyword>Molecular Conformation</keyword><keyword>Molecular Sequence Data</keyword><keyword>Nanostructures/*chemistry/*ultrastructure</keyword><keyword>Nanotechnology/*methods</keyword><keyword>Particle Size</keyword><keyword>Porosity</keyword><keyword>Sequence Analysis, DNA/*methods</keyword><keyword>Surface Properties</keyword></keywords><dates><year>2009</year><pub-dates><date>Apr</date></pub-dates></dates><isbn>1748-3395 (Electronic)
1748-3387 (Linking)</isbn><accession-num>19350039</accession-num><urls><related-urls><url> and is not, as of December 2014, available commercially. The recent distribution of the MinION by Oxford Nanopore Technologies Ltd in an early access programme (named the MinION Access Programme) has made it possible to evaluate the utility of long read sequencing using a device that resembles a large USB memory stick. There were an estimated 26.9 million cases of typhoid fever in 2010 ADDIN EN.CITE <EndNote><Cite><Author>Buckle</Author><Year>2012</Year><RecNum>36</RecNum><DisplayText><style face="superscript">6</style></DisplayText><record><rec-number>36</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">36</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Buckle, G. C.</author><author>Walker, C. L.</author><author>Black, R. E.</author></authors></contributors><auth-address>Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA.</auth-address><titles><title>Typhoid fever and paratyphoid fever: Systematic review to estimate global morbidity and mortality for 2010</title><secondary-title>J Glob Health</secondary-title><alt-title>Journal of global health</alt-title></titles><pages>010401</pages><volume>2</volume><number>1</number><dates><year>2012</year><pub-dates><date>Jun</date></pub-dates></dates><isbn>2047-2986 (Electronic)
2047-2978 (Linking)</isbn><accession-num>23198130</accession-num><urls><related-urls><url> with a very high proportion of those cases in urban slums ADDIN EN.CITE <EndNote><Cite><Author>Wain</Author><Year>2014</Year><RecNum>46</RecNum><DisplayText><style face="superscript">7</style></DisplayText><record><rec-number>46</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">46</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Wain, J</author><author>Hendriksen, RS</author><author>Mikoleit, ML</author><author>Keddy, KH</author><author>Ochiai, RL</author></authors></contributors><titles><title>Typhoid Fever</title><secondary-title>Lancet</secondary-title></titles><volume>In press</volume><dates><year>2014</year></dates><urls></urls></record></Cite></EndNote>7. A recent emergence of a globally distributed multidrug resistant (MDR) Salmonella enterica serovar Typhi (S. Typhi) haplotype, H58, has been observed contributing to a reduction in genetic diversity of extant S. Typhi PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Sb3VtYWduYWM8L0F1dGhvcj48WWVhcj4yMDA2PC9ZZWFy
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ADDIN EN.CITE.DATA 13 encoded on Tn10 and Tn9. The specific resistance plasmid (plasmid PST6 (incHI1)) typical of H58 isolates was, however, not present, raising the possibility that an antibiotic resistance island has integrated into the H58 chromosome.Here, we report a hybrid assembly of combined MinION and Illumina HiSeq data to identify the structure and insertion site of a chromosomal antibiotic resistance island in S. Typhi H58, which, despite many “whole genome” sequencing projects ADDIN EN.CITE <EndNote><Cite><Author>Le</Author><Year>2007</Year><RecNum>17</RecNum><DisplayText><style face="superscript">14</style></DisplayText><record><rec-number>17</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">17</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Le, T. A.</author><author>Fabre, L.</author><author>Roumagnac, P.</author><author>Grimont, P. A.</author><author>Scavizzi, M. R.</author><author>Weill, F. X.</author></authors></contributors><auth-address>Centre National de Reference des Salmonella, Unite de Biodiversite des Bacteries Pathogenes Emergentes, Institut Pasteur, 28 rue du Docteur Roux, 75724, Paris Cedex 15, France.</auth-address><titles><title>Clonal expansion and microevolution of quinolone-resistant Salmonella enterica serotype typhi in Vietnam from 1996 to 2004</title><secondary-title>J Clin Microbiol</secondary-title><alt-title>Journal of clinical microbiology</alt-title></titles><pages>3485-92</pages><volume>45</volume><number>11</number><keywords><keyword>Bacteriophage Typing</keyword><keyword>Drug Resistance, Multiple, Bacterial</keyword><keyword>Electrophoresis, Gel, Pulsed-Field</keyword><keyword>Evolution, Molecular</keyword><keyword>Microbial Sensitivity Tests</keyword><keyword>Polymorphism, Single Nucleotide</keyword><keyword>Quinolones/*pharmacology</keyword><keyword>Ribotyping</keyword><keyword>Salmonella typhi/*classification/drug effects/genetics</keyword><keyword>Time Factors</keyword><keyword>Vietnam</keyword></keywords><dates><year>2007</year><pub-dates><date>Nov</date></pub-dates></dates><isbn>0095-1137 (Print)
0095-1137 (Linking)</isbn><accession-num>17728470</accession-num><urls><related-urls><url> has not been previously characterised.Two S. Typhi H58 strains (H125160566 and 08-0446) were sequenced using the Illumina HiSeq and SNP typing was used to confirm HaplotypePEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Ib2x0PC9BdXRob3I+PFllYXI+MjAwODwvWWVhcj48UmVj
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ADDIN EN.CITE.DATA 12. De novo assembly of Illumina sequence for strain 08-0446 (ENA accession no. ERR668456) resulted in 143 contigs, an N50 of 124 Kbp and average genome coverage of 78x (374 million bases of >Q30 data). De novo assembly of strain H125160566 (ENA accession no. ERR668457) resulted in 86 contigs, an N50 of 154 kbp and average genome coverage of 38x (182 million base pairs of >Q30 data). When these contigs were aligned to pAKUPEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Ib2x0PC9BdXRob3I+PFllYXI+MjAwODwvWWVhcj48UmVj
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ADDIN EN.CITE.DATA 13) using BLAST, only 21 kbp (10%) of the plasmid had matches with significant nucleotide similarity. Similar results were obtained when the H125160566 assembly was compared against pHCM1 incHI1 plasmid (found in S. Typhi CT18) ADDIN EN.CITE <EndNote><Cite><Author>Phan</Author><Year>2009</Year><RecNum>43</RecNum><DisplayText><style face="superscript">15</style></DisplayText><record><rec-number>43</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">43</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Phan, M. D.</author><author>Kidgell, C.</author><author>Nair, S.</author><author>Holt, K. E.</author><author>Turner, A. K.</author><author>Hinds, J.</author><author>Butcher, P.</author><author>Cooke, F. J.</author><author>Thomson, N. R.</author><author>Titball, R.</author><author>Bhutta, Z. A.</author><author>Hasan, R.</author><author>Dougan, G.</author><author>Wain, J.</author></authors></contributors><auth-address>The Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.</auth-address><titles><title>Variation in Salmonella enterica serovar typhi IncHI1 plasmids during the global spread of resistant typhoid fever</title><secondary-title>Antimicrob Agents Chemother</secondary-title><alt-title>Antimicrobial agents and chemotherapy</alt-title></titles><pages>716-27</pages><volume>53</volume><number>2</number><keywords><keyword>DNA, Bacterial/genetics</keyword><keyword>Drug Resistance, Bacterial/*genetics</keyword><keyword>Oligonucleotide Array Sequence Analysis</keyword><keyword>Plasmids/*genetics</keyword><keyword>Polymorphism, Restriction Fragment Length</keyword><keyword>Reverse Transcriptase Polymerase Chain Reaction</keyword><keyword>Salmonella typhi/*genetics</keyword><keyword>Typhoid Fever/*epidemiology/*microbiology</keyword><keyword>Vietnam/epidemiology</keyword></keywords><dates><year>2009</year><pub-dates><date>Feb</date></pub-dates></dates><isbn>1098-6596 (Electronic)
0066-4804 (Linking)</isbn><accession-num>19015365</accession-num><urls><related-urls><url>, which has been hypothesised to be a source of the chromosomally encoded resistance genes in H58.Seven Illumina contigs (>100 bp and totalling 20,974 bp) had nucleotide similarity and query coverage with pAKU >90%, including two with 100% similarity that code for transposable elements IS26 and IS1. In addition, four long contigs (15-65 kbp) had short regions of similarity (<100 bp) with pAKU toward the 5’ or 3’ ends (Supplementary Figure 1). These regions included IS1 inverted repeat sequences, indicating that there were two separate IS1 insertion sites. One of these insertions disrupted the yidA gene, while the other was in an intergenic region between STY3618 and STY3619 of the CT18 reference genome. While it is possible to determine whether an insertion site is occupied using traditional methods (PCR), it was not feasible in this case as we did not know the internal structure of the island and it was too large to span by PCR. S. Typhi strain H125160566 was also sequenced on the MinION device for 18 hours resulting in 16,401 sequencing reads (ENA accession no. ERR668747 – the MinION fast5 files contain both raw ‘squiggle plot’ data and associated base-called data) with median length 5,412 bp, a maximum length of 66,748 bp, median Phred score of 5, median accuracy of 68.4% (derived from Phred score) and a total of 93.4 Mbp of sequence data (Table 1A). MinION sequencing produces three types of read for each DNA molecule analysed, template (lead DNA strand), complement (complementary DNA strand) and two-direction (2D). When both template and complement data are available, an additional 2D basecall is performed, providing a consensus. There were 8,209 template, 4,454 complement and 3,738 2D reads. On average, the complement reads were shorter (median 4,728 bp) than the template and two-directional reads (5,614 bp & 5,943 bp respectively) (Supplementary Figure 2). According to the MinON basecalling software (Metrichor version 1.3.1) Phred scores (a measure of the likely accuracy of the base called data), the two direction reads had the highest mean accuracy (83.6%), followed by the complement reads (55.6%) and the template reads (53.9%). Two additional MinION runs were carried out, one was a repeat of H125160566 with overnight (rather than 30 min) library incubation (as recommended by Oxford Nanopore after some reagent problems caused by shipping conditions – ENA accession no. ERR668746) and one with the second S. Typhi H58 isolate (strain 08-04776 – ENA accession no. ERR668983). The data from all three runs were very similar (see Supplementary Table 1). All S. Typhi strain H125160566 MinION reads were mapped to the Illumina assembly of the same strain using the LAST sequence alignment tool ADDIN EN.CITE <EndNote><Cite><Author>Frith</Author><Year>2010</Year><RecNum>142</RecNum><DisplayText><style face="superscript">16</style></DisplayText><record><rec-number>142</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">142</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Frith, M. C.</author><author>Hamada, M.</author><author>Horton, P.</author></authors></contributors><auth-address>Computational Biology Research Center, Institute for Advanced Industrial Science and Technology, Tokyo 135-0064, Japan. martin@cbrc.jp</auth-address><titles><title>Parameters for accurate genome alignment</title><secondary-title>BMC Bioinformatics</secondary-title><alt-title>BMC bioinformatics</alt-title></titles><pages>80</pages><volume>11</volume><edition>2010/02/11</edition><keywords><keyword>Base Sequence</keyword><keyword>Computational Biology/methods</keyword><keyword>*Genome</keyword><keyword>Molecular Sequence Data</keyword><keyword>Proteins/chemistry</keyword><keyword>RNA/chemistry</keyword><keyword>Sequence Alignment/*methods</keyword><keyword>Sequence Analysis, RNA</keyword></keywords><dates><year>2010</year></dates><isbn>1471-2105 (Electronic)
1471-2105 (Linking)</isbn><accession-num>20144198</accession-num><work-type>Research Support, Non-U.S. Gov't</work-type><urls><related-urls><url> (Table 1B). In our hands LAST performed better than other programmes tested (BLAST and BWA mem) as it can align sequences with many mismatches and gaps, which is typical of the sequence data currently produced by the MinION. In total 68.7% (11,278/16,401) of the reads mapped at least once to the Illumina assembly, with 16,337 non-duplicate alignments giving an average coverage of 14x. Mismatches between MinION reads and the Illumina assembly were identified, giving a mean percentage accuracy for all read types of 65.6%. Complement reads had the lowest similarity (61.6%), followed by template (64.3%) then two-directional reads (71.5%). There were a large number of gaps in the alignments, with a median of 18.5% of the length of alignments consisting of paring the mapping derived accuracy of MinION reads with the Phred derived accuracy of the same reads provided insight into the reliability of Phred calling for MinION data. For H125160566, the template and complement reads had median mapping derived accuracies of 64.3% and 61.6% while the median Phred derived accuracy for these same reads was 60.2%, demonstrating the reliability of Phred scores for these read types. For the 2D reads, however, the median mapping accuracy was 71.5% while the Phred derived accuracy was 84.2%, indicating that the Phred scoring algorithm overestimated the accuracy of these reads. The error profile of the MinION H125160566 data set was characterised. In the 70.8 megabases of aligned nanopore sequence an indel occurred every 5.9 bases on average. Two-thirds of these indels were deletions (8.6 Mbp) while one third were insertions (4.15 Mbp). The mean deletion length was 1.7 bp, while the mean insertion length was 1.6 bp. Both insertion and deletion lengths had a negative exponential distribution (Supplementary Figures 3 & 4).A z-score was calculated to identify k-mers that were deleted from mapped MinION reads at a higher than expected frequency. When z-scores for deletions of 3 to 6 bp were binned by GC content, the tendency of the MinION to skip k-mers of low purine-pyrimidine diversity could be seen (Figure 1). In particular, there were two 3-mers with a z-score greater than 3, GGG (z = 3.3) and CCC (z = 3.2), two 4-mers with a z-score greater than 3, GGGG (z = 4.4) and CCCC (z = 4) and four 5-mers with a z-score > 4, AAAAA, TTTTT, CCCCC and GGGGG. Two more diverse k-mers that had high z-scores were TAGGCA and TAGGGC, with z-scores of 9.6 and 9.5 respectively. When insertions were examined there were two 3-mers with a z-score higher than 3, CCC (z = 3.7) and GGG (z = 3.6) and two 4-mers with a z-score higher than 3, CCCC (z = 6.1) and GGGG (z = 6.0).To identify substitutions, all non-consensus bases were extracted from the MinION alignment to the Illumina assembly. This showed that A to T and T to A substitutions were approximately half as frequent as other substitutions (Supplementary Table 2). These data suggest that MinION currently has difficulty differentiating Gs and Cs, particularly when these bases are present in homopolymeric tracts.We next used the long MinION reads to scaffold the H125160566 Illumina contigs with homology to pAKU (see methods section) in order to determine the structure and chromosomal insertion site of the antibiotic resistance island. Forty MinION reads that were informative as to the structure and insertion site of the island were identified and these reads were used to link the island contigs across the insertion sequences (Supplementary Figure 1). There were between two and four reads spanning each break in the island assembly.The breaks in the Illumina assembly were caused by the presence of insertion sequences in all but two cases. These additional breaks were caused by a decrease in Illumina data coverage (from an average of 38x to 1-2x) in the merA and hyp genes, resulting in 1.3 Kb and 0.3Kb fragments missing in the Illumina assembly. This low coverage was associated with high GC content (>70%), a known problem with Nextera Illumina sequencing ADDIN EN.CITE <EndNote><Cite><Author>Adey</Author><Year>2010</Year><RecNum>48</RecNum><DisplayText><style face="superscript">17</style></DisplayText><record><rec-number>48</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">48</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Adey, A.</author><author>Morrison, H. G.</author><author>Asan,</author><author>Xun, X.</author><author>Kitzman, J. O.</author><author>Turner, E. H.</author><author>Stackhouse, B.</author><author>MacKenzie, A. P.</author><author>Caruccio, N. C.</author><author>Zhang, X.</author><author>Shendure, J.</author></authors></contributors><auth-address>Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA. acadey@uw.edu</auth-address><titles><title>Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition</title><secondary-title>Genome Biol</secondary-title><alt-title>Genome biology</alt-title></titles><pages>R119</pages><volume>11</volume><number>12</number><keywords><keyword>Animals</keyword><keyword>DNA/isolation & purification</keyword><keyword>*DNA Fragmentation</keyword><keyword>Drosophila/genetics</keyword><keyword>Escherichia coli/genetics</keyword><keyword>Exons</keyword><keyword>Genome, Human</keyword><keyword>Genome, Insect</keyword><keyword>*Genomic Library</keyword><keyword>Humans</keyword><keyword>Polymerase Chain Reaction/*methods</keyword><keyword>Sequence Analysis, DNA/*methods</keyword><keyword>Transposases/metabolism</keyword></keywords><dates><year>2010</year></dates><isbn>1465-6914 (Electronic)
1465-6906 (Linking)</isbn><accession-num>21143862</accession-num><urls><related-urls><url>. When determining the island sequence in these regions, data was derived from confirmatory PCR products rather than the erroneous Illumina-only de novo assembly data. The MinION reads identified the insertion site as the yidA gene (Supplementary Figure 5). Mapping from both ends of this disrupted gene into the island showed that it is flanked by IS1 elements. The island contains several of the resistance genes/elements found on IncHI1 plasmids in MDR S. Typhi including strA, strB, sulI, sulII and blaTEM-1 (Figure 2 – ENA accession no. PRJEB7681). The structure and insertion site of the island were confirmed by PCR followed by Sanger sequencing (see methods section). This confirmation was not possible until the island structure was solved (using the hybrid assembly) because of the low-coverage and misassembled Illumina data.The second H58 S.Typhi strain analysed in this study (08-04776) contained the antibiotic resistance island and an IncN plasmid. The Illumina contigs from 08-04776 contained one 7 kb contig with a dihydopteroate synthase gene (sulI) and there were no other sulI genes in the assembly. When MinION reads were mapped to this contig, they did not map contiguously from this contig to other island contigs, but rather, mapped only to the sulI encoding section. Therefore, we hypothesise that 08-04776 encodes two sulI genes; one on the IncN plasmid and one on the chromosomally inserted island. The assembly of the Illumina data collapsed these two genes into a single copy, which represented a misassembly that confounded analysis and was only resolved using the MinION reads.In order to demonstrate the utility of MinION sequence for hybrid genome assembly, the H125160566 strain Illumina and Minion data were assembled using SPAdes ADDIN EN.CITE <EndNote><Cite><Author>Bankevich</Author><Year>2012</Year><RecNum>144</RecNum><DisplayText><style face="superscript">18</style></DisplayText><record><rec-number>144</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">144</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Bankevich, A.</author><author>Nurk, S.</author><author>Antipov, D.</author><author>Gurevich, A. A.</author><author>Dvorkin, M.</author><author>Kulikov, A. S.</author><author>Lesin, V. M.</author><author>Nikolenko, S. I.</author><author>Pham, S.</author><author>Prjibelski, A. D.</author><author>Pyshkin, A. V.</author><author>Sirotkin, A. V.</author><author>Vyahhi, N.</author><author>Tesler, G.</author><author>Alekseyev, M. A.</author><author>Pevzner, P. A.</author></authors></contributors><auth-address>Algorithmic Biology Laboratory, St. Petersburg Academic University, Russian Academy of Sciences, St. Petersburg, Russia.</auth-address><titles><title>SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing</title><secondary-title>J Comput Biol</secondary-title><alt-title>Journal of computational biology : a journal of computational molecular cell biology</alt-title></titles><pages>455-77</pages><volume>19</volume><number>5</number><keywords><keyword>*Algorithms</keyword><keyword>Bacteria/*genetics</keyword><keyword>*Genome, Bacterial</keyword><keyword>Metagenomics/*methods</keyword><keyword>Sequence Analysis, DNA/methods</keyword><keyword>Single-Cell Analysis/*methods</keyword></keywords><dates><year>2012</year><pub-dates><date>May</date></pub-dates></dates><isbn>1557-8666 (Electronic)
1066-5277 (Linking)</isbn><accession-num>22506599</accession-num><urls><related-urls><url>. This method resolved the genome into 34 contigs, with an N50 of 319kbp, whereas the Illumina-only assembly produced 86 contigs with an N50 of 154kbp. The SPAdes hybrid assembly confirmed the yidA insertion site but failed to resolve the complete island structure with contig breaks in the hyp and merA genes, presumably due to the low coverage regions in the Illumina data. SPAdes hybrid assembly demonstrated significant improvement over Illumina-only assembly - further improvements would be possible given higher coverage MinION data. To test the hypothesis that the antibiotic resistance island originated from an S. Typhi MDR IncHI1 plasmid we compared it to pHCM1 and other publicly deposited sequences using BLASTN – surprisingly one of the nearest matches (88% coverage, 99% identity) was pRSB107, a plasmid that was sequenced in sewagePEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TemN6ZXBhbm93c2tpPC9BdXRob3I+PFllYXI+MjAwNTwv
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ADDIN EN.CITE.DATA 13 and now, as we have demonstrated, chromosomal integration of a set of genes. These genes have no obvious source and the similarity of the island with a plasmid identified in sewage has opened up our research into its origins. Acquisition of this antibiotic resistance island has allowed a clonal sweep within the population of one of the classic human pathogens.If the enormous potential of applying WGS in healthcare is to be realised, then public health and clinical laboratories must have easy access to technology capable of providing fully assembled bacterial genomes. This will enable bacterial identification, sub-typing and resistance gene detection at the point of clinical need. MinION technology demonstrates potential for such applications as it is highly accessible and is already well supported by the bioinformatics community, with two software packages, poretools and poRe, released for data analysis PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5XYXRzb248L0F1dGhvcj48WWVhcj4yMDE0PC9ZZWFyPjxS
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ADDIN EN.CITE.DATA 20, 21. In our hands MinION flow cells with >400 active nanopores (out of a total of 512) produce yields up to 150 Mbp in 48 hours (max yield in this study was 90Mbp) with an accuracy of ~72% for 2D reads. This is comparable to R7 MinION data published by Quick et al. although the yield they achieved was approx. 400Mb and the mean accuracy for 2D reads was higher (approx. 80%) ADDIN EN.CITE <EndNote><Cite><Author>Quick</Author><Year>2014</Year><RecNum>158</RecNum><DisplayText><style face="superscript">22</style></DisplayText><record><rec-number>158</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">158</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Quick, J.</author><author>Quinlan, A. R.</author><author>Loman, N. J.</author></authors></contributors><auth-address>Institute of Microbiology and Infection, University of Birmingham, Birmingham B15 2TT, UK ; NIHR Surgical Reconstruction and Microbiology Research Centre, University of Birmingham, Birmingham B15 2TT, UK.
Center for Public Health Genomics, University of Virginia, Charlottesville VA 22908, USA.
Institute of Microbiology and Infection, University of Birmingham, Birmingham B15 2TT, UK.</auth-address><titles><title>A reference bacterial genome dataset generated on the MinION portable single-molecule nanopore sequencer</title><secondary-title>Gigascience</secondary-title><alt-title>GigaScience</alt-title></titles><periodical><full-title>Gigascience</full-title><abbr-1>GigaScience</abbr-1></periodical><alt-periodical><full-title>Gigascience</full-title><abbr-1>GigaScience</abbr-1></alt-periodical><pages>22</pages><volume>3</volume><edition>2014/11/12</edition><dates><year>2014</year></dates><isbn>2047-217X (Electronic)
2047-217X (Linking)</isbn><accession-num>25386338</accession-num><urls><related-urls><url>. These differences in performance are most likely related to variations in the quality of the flow cells. These data are a significant improvement on the 10% accuracy reported in an early MinION paper by Mikheyev et al ADDIN EN.CITE <EndNote><Cite><Author>Mikheyev</Author><Year>2014</Year><RecNum>141</RecNum><DisplayText><style face="superscript">23</style></DisplayText><record><rec-number>141</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">141</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Mikheyev, Alexander S.</author><author>Tin, Mandy M. Y.</author></authors></contributors><titles><title>A first look at the Oxford Nanopore MinION sequencer</title><secondary-title>Molecular Ecology Resources</secondary-title></titles><pages>n/a-n/a</pages><keywords><keyword>genomics</keyword><keyword>next-generation sequencing</keyword><keyword>nanopore sequencing</keyword><keyword>bioinformatics</keyword></keywords><dates><year>2014</year></dates><isbn>1755-0998</isbn><urls><related-urls><url>. We did not make a comparison with other long read technologies such as PacBio, as this was not the purpose of our study, but the median and maximum MinION read lengths (approx. 6 Kbp and 60 Kbp respectively) were comparable to those reported for recent PacBio chemistry (approx. 7-12 Kbp average and 20-30 Kbp maximum length filtered subreads) although the yield (approx. 90 Mbp for MinION vs 350 Mbp for PacBio) and accuracy (approx. 72% vs 85%) were lower ADDIN EN.CITE <EndNote><Cite><Author>Kim</Author><Year>2014</Year><RecNum>155</RecNum><DisplayText><style face="superscript">24</style></DisplayText><record><rec-number>155</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">155</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Kim, Kristi E</author><author>Peluso, Paul</author><author>Baybayan, Primo</author><author>Yeadon, Patricia Jane</author><author>Yu, Charles</author><author>Fisher, William</author><author>Chin, Chen-Shan</author><author>Rapicavoli, Nicole A</author><author>Rank, David R</author><author>Li, Joachim</author><author>Catcheside, David</author><author>Celniker, Susan E</author><author>Phillippy, Adam M</author><author>Bergman, Casey M</author><author>Landolin, Jane M</author></authors></contributors><titles><title>Long-read, whole genome shotgun sequence data for five model organisms</title><secondary-title>Biorxiv</secondary-title></titles><pages> 00:00:00</date></pub-dates></dates><work-type>Journal Article</work-type><urls><related-urls><url>. It must be noted that, in our hands, the length of the input DNA, not the chemistry, determined read length on the MinION and we did not attempt to maximise read length.Further improvements are needed in MinION data quality and yield, which are promised in flow cell and library preparation kit upgrades. The recent R7.3 flow cell and SQK-MAP003 gDNA library preparation kit have increased the proportion of full 2D reads (best quality 2D reads) from 8% to 86% ADDIN EN.CITE <EndNote><Cite><Author>Quick</Author><Year>2014</Year><RecNum>158</RecNum><DisplayText><style face="superscript">22</style></DisplayText><record><rec-number>158</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">158</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Quick, J.</author><author>Quinlan, A. R.</author><author>Loman, N. J.</author></authors></contributors><auth-address>Institute of Microbiology and Infection, University of Birmingham, Birmingham B15 2TT, UK ; NIHR Surgical Reconstruction and Microbiology Research Centre, University of Birmingham, Birmingham B15 2TT, UK.
Center for Public Health Genomics, University of Virginia, Charlottesville VA 22908, USA.
Institute of Microbiology and Infection, University of Birmingham, Birmingham B15 2TT, UK.</auth-address><titles><title>A reference bacterial genome dataset generated on the MinION portable single-molecule nanopore sequencer</title><secondary-title>Gigascience</secondary-title><alt-title>GigaScience</alt-title></titles><periodical><full-title>Gigascience</full-title><abbr-1>GigaScience</abbr-1></periodical><alt-periodical><full-title>Gigascience</full-title><abbr-1>GigaScience</abbr-1></alt-periodical><pages>22</pages><volume>3</volume><edition>2014/11/12</edition><dates><year>2014</year></dates><isbn>2047-217X (Electronic)
2047-217X (Linking)</isbn><accession-num>25386338</accession-num><urls><related-urls><url> which results in improved accuracy (approx. 85% for full 2D reads). Recent bioinformatic developments have allowed the de novo assembly of genomes from high coverage (~100x) PacBio data ADDIN EN.CITE <EndNote><Cite><Author>Chin</Author><Year>2013</Year><RecNum>49</RecNum><DisplayText><style face="superscript">25</style></DisplayText><record><rec-number>49</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">49</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Chin, C. S.</author><author>Alexander, D. H.</author><author>Marks, P.</author><author>Klammer, A. A.</author><author>Drake, J.</author><author>Heiner, C.</author><author>Clum, A.</author><author>Copeland, A.</author><author>Huddleston, J.</author><author>Eichler, E. E.</author><author>Turner, S. W.</author><author>Korlach, J.</author></authors></contributors><auth-address>Pacific Biosciences, Menlo Park, California, USA.</auth-address><titles><title>Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data</title><secondary-title>Nat Methods</secondary-title><alt-title>Nature methods</alt-title></titles><pages>563-9</pages><volume>10</volume><number>6</number><keywords><keyword>Chromosomes, Artificial, Bacterial</keyword><keyword>Escherichia coli/genetics</keyword><keyword>Gene Library</keyword><keyword>*Genome, Bacterial</keyword><keyword>Humans</keyword><keyword>Repetitive Sequences, Nucleic Acid</keyword><keyword>Sequence Analysis, DNA/*methods</keyword></keywords><dates><year>2013</year><pub-dates><date>Jun</date></pub-dates></dates><isbn>1548-7105 (Electronic)
1548-7091 (Linking)</isbn><accession-num>23644548</accession-num><urls><related-urls><url> and this may also be possible using improved MinION technology in the future. In conclusion, the MinION data presented here has a higher error rate than other widely used long-read and short-read sequencing platforms. However, the reads produced are of comparable length to PacBio and using ‘off the shelf’ bioinformatic tools, we have solved the structure of a complex antibiotic resistance island. Nanopore technology has the potential to create a paradigm shift in genomics; low cost, long read sequencing can be carried out in non-specialist laboratories, both research and service. The question is: can nanopore sequencing replace short read sequencing technology? The answer: not yet, but it is knocking at the door.AcknowledgementsMartin Day (Salmonella Reference Unit, Public Health England) for antibiotic susceptibility tests. JOG and JW were funded by the University of East Anglia. We would like to thank Oxford Nanopore Technologies Ltd. for including us in the MinION Access Programme. We would also like to thank Lex Nederbragt for a thorough review and contributions toward the presentation of Figure peting financial interestsJ.O’G is a participant of Oxford Nanopore’s MinION Access Programme (MAP) and received the MinION device and flowcells used for this study free of charge.Author contributionsP.M.A, S.N, T.D, J.W and J.O’G conceived the study, performed the analysis and wrote the first draft of the manuscript. J.O’G and S.M performed the MinION sequencing. P.M.A and T.D performed the bioinformatics analysis. S.N performed the PCR analysis and co-ordinated the Illumina sequencing. P.M.A, T.D, S.R, W.R, J.W and J.O’G analysed the resistance island structure and insertion site and devised the figures. All authors contributed to editing and data analysis of the final manuscript.TablesTable 1: (A) Read statistics for a single 18 hour MinION run of S. Typhi H125160566, broken down by read type (B) Statistics on mapping of reads to the H125160566 Illumina assembly(A) Read stats?Number of readsMedian length (bp)Max length (bp)Total length (Mbp)Median Phred scoreMedian AccuracyTotal1640154126674893.4568.40% Template820956145881049.6349.90% Complement445447286674821.1460.20% 2D373859433163022.7884.20%(B) Mapping stats?Number reads alignedNumber alignmentsTotal length alignments (Mbp)Median accuracyMedian gapsTotal112781633770.864.2%17.8% Template4705619431.464.9%17.1% Complement3171527517.461.1%23.5% 2D3402486822.072.6%14.8%REFERENCES ADDIN EN.REFLIST 1.Underwood, A.P. et al. Public health value of next-generation DNA sequencing of enterohemorrhagic Escherichia coli isolates from an outbreak. J Clin Microbiol 51, 232-237 (2013).2.Wain, J. & Mavrogiorgou, E. Next-generation sequencing in clinical microbiology. Expert Rev Mol Diagn 13, 225-227 (2013).3.Thomson, N. et al. 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Typhi strain 08-04776 was isolated in 2008 at the Robert Koch Institute, Wernigerode, from a patient returning from Iraq. The sequenced S. Typhi strain CT18PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5QYXJraGlsbDwvQXV0aG9yPjxZZWFyPjIwMDE8L1llYXI+
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ADDIN EN.CITE.DATA 26 and S. Paratyphi A strain AKU 12601 ADDIN EN.CITE <EndNote><Cite><Author>Holt</Author><Year>2009</Year><RecNum>52</RecNum><DisplayText><style face="superscript">27</style></DisplayText><record><rec-number>52</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">52</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Holt, K. E.</author><author>Thomson, N. R.</author><author>Wain, J.</author><author>Langridge, G. C.</author><author>Hasan, R.</author><author>Bhutta, Z. A.</author><author>Quail, M. A.</author><author>Norbertczak, H.</author><author>Walker, D.</author><author>Simmonds, M.</author><author>White, B.</author><author>Bason, N.</author><author>Mungall, K.</author><author>Dougan, G.</author><author>Parkhill, J.</author></authors></contributors><auth-address>Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK. kh2@sanger.ac.uk</auth-address><titles><title>Pseudogene accumulation in the evolutionary histories of Salmonella enterica serovars Paratyphi A and Typhi</title><secondary-title>BMC Genomics</secondary-title><alt-title>BMC genomics</alt-title></titles><pages>36</pages><volume>10</volume><keywords><keyword>DNA, Bacterial/genetics</keyword><keyword>Evolution, Molecular</keyword><keyword>Genes, Bacterial</keyword><keyword>*Genome, Bacterial</keyword><keyword>Phylogeny</keyword><keyword>*Pseudogenes</keyword><keyword>Recombination, Genetic</keyword><keyword>Salmonella paratyphi A/*genetics</keyword><keyword>Salmonella typhi/*genetics</keyword><keyword>Sequence Analysis, DNA</keyword></keywords><dates><year>2009</year></dates><isbn>1471-2164 (Electronic)
1471-2164 (Linking)</isbn><accession-num>19159446</accession-num><urls><related-urls><url> were used as controls in the PCR experiments. The isolates were biochemically typed and serotyped in accordance to the White-Kauffmann-le Minor Scheme ADDIN EN.CITE <EndNote><Cite><Author>Grimont</Author><Year>2007</Year><RecNum>70</RecNum><DisplayText><style face="superscript">28</style></DisplayText><record><rec-number>70</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">70</key></foreign-keys><ref-type name="Report">27</ref-type><contributors><authors><author>Grimont, AD</author><author>Weill, FX</author></authors></contributors><titles><title>Antigenic formulae of the Salmonella serovars</title></titles><volume>9th edition</volume><dates><year>2007</year></dates><pub-location>Geneva</pub-location><publisher>World Health Organisation</publisher><urls></urls></record></Cite></EndNote>28.Phage TypingS. Typhi strains were typed according to the phage-typing scheme described by Callow,1959 ADDIN EN.CITE <EndNote><Cite><Author>Callow</Author><Year>1959</Year><RecNum>72</RecNum><DisplayText><style face="superscript">29</style></DisplayText><record><rec-number>72</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">72</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Callow, BR</author></authors></contributors><titles><title>A new phage-typing scheme for Salmonella Typhimurium</title><secondary-title>Journal of Hygiene</secondary-title></titles><pages>346-359</pages><volume>57</volume><dates><year>1959</year></dates><urls></urls></record></Cite></EndNote>29.Antimicrobial resistanceS. Typhi strains were tested for resistance to antimicrobials using standard disc diffusion methods for Enterobacteriaceae in accordance to the European Committee on Antimicrobial Susceptibility Testing (EUCAST, Version 3.1) guidelines. The control strain used was Escherichia coli ATCC 25922.DNA extraction and quantificationChromosomal DNA used for PCR and sequencing was isolated using the Wizard genomic DNA purification kit (Promega) according to the manufacturer’s instructions.DNA was quantified using the Quant-iT? dsDNA High Sensitivity Assay (HS) Kit (Life Technologies) which enables high-throughput quantitation of samples on the GloMax? Multi+ Detection System (Promega).Illumina DNA sequencingExtracted DNA was prepared using the NexteraXT library preparation method according to the manufacturer’s instructions and sequenced with a standard 2 x 101 base protocol on a HiSeq Instrument (Illumina, San Diego).Illumina data analysisS.Typhi strains were confirmed as being Haplotype 58 from the Illumina sequencing data by mapping to the S. Typhi CT18 reference genome (NC003198) and determining the presence of haplotype 58 specific SNPs ADDIN EN.CITE <EndNote><Cite><Author>Holt</Author><Year>2008</Year><RecNum>156</RecNum><DisplayText><style face="superscript">30</style></DisplayText><record><rec-number>156</rec-number><foreign-keys><key app="EN" db-id="xtaz9ar9t59f5her5v8p0v5vaaf0rfw902zf">156</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Holt, Kathryn E.</author><author>Parkhill, Julian</author><author>Mazzoni, Camila J.</author><author>Roumagnac, Philippe</author><author>Weill, Francois-Xavier</author><author>Goodhead, Ian</author><author>Rance, Richard</author><author>Baker, Stephen</author><author>Maskell, Duncan J.</author><author>Wain, John</author><author>Dolecek, Christiane</author><author>Achtman, Mark</author><author>Dougan, Gordon</author></authors></contributors><titles><title>High-throughput sequencing provides insights into genome variation and evolution in Salmonella Typhi</title><secondary-title>Nat Genet</secondary-title></titles><periodical><full-title>Nat Genet</full-title></periodical><pages>987-993</pages><volume>40</volume><number>8</number><dates><year>2008</year></dates><publisher>Nature Publishing Group</publisher><isbn>1061-4036</isbn><work-type>10.1038/ng.195</work-type><urls><related-urls><url> reads were assembled using SPAdes v3.1.1 ADDIN EN.CITE <EndNote><Cite><Author>Bankevich</Author><Year>2012</Year><RecNum>144</RecNum><DisplayText><style face="superscript">18</style></DisplayText><record><rec-number>144</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">144</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Bankevich, A.</author><author>Nurk, S.</author><author>Antipov, D.</author><author>Gurevich, A. A.</author><author>Dvorkin, M.</author><author>Kulikov, A. S.</author><author>Lesin, V. M.</author><author>Nikolenko, S. I.</author><author>Pham, S.</author><author>Prjibelski, A. D.</author><author>Pyshkin, A. V.</author><author>Sirotkin, A. V.</author><author>Vyahhi, N.</author><author>Tesler, G.</author><author>Alekseyev, M. A.</author><author>Pevzner, P. A.</author></authors></contributors><auth-address>Algorithmic Biology Laboratory, St. Petersburg Academic University, Russian Academy of Sciences, St. Petersburg, Russia.</auth-address><titles><title>SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing</title><secondary-title>J Comput Biol</secondary-title><alt-title>Journal of computational biology : a journal of computational molecular cell biology</alt-title></titles><pages>455-77</pages><volume>19</volume><number>5</number><keywords><keyword>*Algorithms</keyword><keyword>Bacteria/*genetics</keyword><keyword>*Genome, Bacterial</keyword><keyword>Metagenomics/*methods</keyword><keyword>Sequence Analysis, DNA/methods</keyword><keyword>Single-Cell Analysis/*methods</keyword></keywords><dates><year>2012</year><pub-dates><date>May</date></pub-dates></dates><isbn>1557-8666 (Electronic)
1066-5277 (Linking)</isbn><accession-num>22506599</accession-num><urls><related-urls><url> with the ‘—careful’ flag and kmers of 21, 33, 55 and 77. The resulting contigs were compared against the IncHI1 plasmid pAKU (NCBI accession AM412236) using BLAST within the BioPython framework ADDIN EN.CITE <EndNote><Cite><Author>Cock</Author><Year>2009</Year><RecNum>24</RecNum><DisplayText><style face="superscript">31</style></DisplayText><record><rec-number>24</rec-number><foreign-keys><key app="EN" db-id="etv0xzxs0rvef0ef5z95xf0pzrfzs0x5rva9">24</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Cock, P. J.</author><author>Antao, T.</author><author>Chang, J. T.</author><author>Chapman, B. A.</author><author>Cox, C. J.</author><author>Dalke, A.</author><author>Friedberg, I.</author><author>Hamelryck, T.</author><author>Kauff, F.</author><author>Wilczynski, B.</author><author>de Hoon, M. J.</author></authors></contributors><auth-address>Plant Pathology, SCRI, Invergowrie, Dundee, UK. peter.cock@scri.ac.uk</auth-address><titles><title>Biopython: freely available Python tools for computational molecular biology and bioinformatics</title><secondary-title>Bioinformatics</secondary-title><alt-title>Bioinformatics</alt-title></titles><periodical><full-title>Bioinformatics</full-title><abbr-1>Bioinformatics</abbr-1></periodical><alt-periodical><full-title>Bioinformatics</full-title><abbr-1>Bioinformatics</abbr-1></alt-periodical><pages>1422-3</pages><volume>25</volume><number>11</number><keywords><keyword>Computational Biology/*methods</keyword><keyword>Databases, Factual</keyword><keyword>Internet</keyword><keyword>Programming Languages</keyword><keyword>*Software</keyword></keywords><dates><year>2009</year><pub-dates><date>Jun 1</date></pub-dates></dates><isbn>1367-4811 (Electronic)
1367-4803 (Linking)</isbn><accession-num>19304878</accession-num><urls><related-urls><url> and all contigs that contained a region that matched pAKU with an E-value of less than 1 x 10-20 were extracted from the assembly. The alternative IncHI1 plasmid pHCM1 (NCBI accession AL513383) was also used as the reference and the same regions of the same contigs matched the plasmid. MinION library preparationThe Oxford Nanopore MinION Genomic DNA Sequencing Kit was used to prepare the S. Typhi DNA libraries according to the manufacturer’s instructions. Briefly, kit reagents were either thawed on ice (tether, DNA CS, HP adapter, adapter mix, ligation buffer, HP motor and fuel mix) or at room temperature (wash buffer, elution buffer and EP buffer) and once thawed, all reagent were kept on ice until required. One microgram of DNA was diluted to 85μl in molecular grade water and sheared using a Covaris g-TUBE according to the manufacturer’s instructions by centrifuging through the column in both directions at 3,600 x g for 60 sec (sample was pulse centrifuged at 4,000 x g if all liquid hadn’t passed through the column). End-repair was performed on the sheared DNA using NEBNext End Repair Module (New England Biolabs cat. no. E6050) by adding 10?l reaction buffer and 5?l enzyme mix and incubating for 30 minutes at 20°C. The end-repaired DNA was then purified using 1 x volume (100?l) Agencourt AMPure XP beads (Beckman Coulter Inc cat. no. A63880) according to the manufacturer’s instructions. Samples were incubated on a magnetic rack (Invitrogen MagnaRack cat. no. CS15000) for three minutes and washed twice in 200?l 70% ethanol while still on the magnet. DNA was eluted in 25.2?l of molecular grade water. dA-tailing was then performed using NEBNext dA-tailing module (New England Biolabs cat. no. E6053) in a total of 30?l by adding 3?l buffer and 1.8?l Klenow Fragment to the clean DNA. The sample was incubated in a thermal cycler for 30 minutes at 37°C. A ligation reaction was then performed by adding the following reagents in order: 50?l dA-tailed DNA; 10?l adapter mix; 10?l HP adapter; 50?l Blunt/TA DNA ligase (New England Biolabs M0367). The reaction was gently pipetted up and down to mix and incubated at room temperature for 10 minutes. The adapted-ligated DNA was purified using 0.4 x volume (40?l) Agencourt AMPure XP beads according to the manufacturer’s instructions, but using the Oxford Nanopore-supplied wash buffer (150?l x one wash only making sure to remove all wash buffer) and elution buffer (25?l). The tether was annealed by adding 10?l tether to the 25?l ligated DNA and incubating for 10 minutes at room temperature. Finally, 15?l HP motor was added to the reaction (50?l total volume) and incubated for 30 minutes (or overnight) at room temperature. The library was then ready for sequencing on the MinION (see below). MinION sequencing During the last half-hour library preparation incubation the MinION was connected to the computer (a PC that met the requirements for running the MinION and associated software: Windows 7; USB3; SSD; i7 processor) via USB3; the MinION flow cell (R7 flow cell chemistry) was removed from its packaging and inserted into the MinION; the MinKNOW software was opened; a flow cell quality control programme was run to assess pore activity; and the flow cell was equilibrated by pipetting two aliquots of EP buffer into the flow cell, incubating for 10 minutes after each addition. Immediately after the library preparation reaction was complete, 6?l library, 140?l EP buffer and 4?l fuel mix were combined in a fresh tube, mixed by gentle pipetting and loaded onto the MinION according to the manufacturer’s instructions. The 48-hour sequencing protocol was chosen and the sequencing reaction was started.MinION data analysisOnce the sequencing run had begun, the Metrichor programme was started and the MinION automatically uploads resulting raw data to the Metrichor platform for base calling (workflow r7 2D Basecalling rev 1.3.1), although all data required for base calling are available to the user. The base calling via Metrichor returns a series of ‘fast5’ files, which contain, among other things; ‘events’ (i.e. perturbations of current through the pore) and sequence data in fastq format with associated quality scores. Percentage accuracy of the reads was calculated from the phred score ADDIN EN.CITE <EndNote><Cite><Author>Ewing</Author><Year>1998</Year><RecNum>25</RecNum><DisplayText><style face="superscript">32</style></DisplayText><record><rec-number>25</rec-number><foreign-keys><key app="EN" db-id="etv0xzxs0rvef0ef5z95xf0pzrfzs0x5rva9">25</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Ewing, B.</author><author>Green, P.</author></authors></contributors><auth-address>Department of Molecular Biotechnology, University of Washington, Seattle, Washington 98195-7730, USA.</auth-address><titles><title>Base-calling of automated sequencer traces using phred. II. Error probabilities</title><secondary-title>Genome Res</secondary-title><alt-title>Genome research</alt-title></titles><periodical><full-title>Genome Res</full-title><abbr-1>Genome research</abbr-1></periodical><alt-periodical><full-title>Genome Res</full-title><abbr-1>Genome research</abbr-1></alt-periodical><pages>186-94</pages><volume>8</volume><number>3</number><keywords><keyword>Base Sequence</keyword><keyword>Chimera</keyword><keyword>Cloning, Molecular/methods</keyword><keyword>Data Interpretation, Statistical</keyword><keyword>Discriminant Analysis</keyword><keyword>Genetic Vectors</keyword><keyword>Human Genome Project</keyword><keyword>Humans</keyword><keyword>Probability</keyword><keyword>Quality Control</keyword><keyword>Reproducibility of Results</keyword><keyword>Sequence Analysis, DNA/*methods/standards/*statistics & numerical data</keyword><keyword>*Software</keyword></keywords><dates><year>1998</year><pub-dates><date>Mar</date></pub-dates></dates><isbn>1088-9051 (Print)
1088-9051 (Linking)</isbn><accession-num>9521922</accession-num><urls><related-urls><url>. The fast5 data is stored in Hierarchical Data Format v5 (HDF5), which can be viewed via using a graphic user interface application such as HDFView () or using a package such as h5py for Python (). Fast5 files from Metrichor were parsed and the sequence extracted in fasta and fastq format using the Poretools v 0.3.0 library ADDIN EN.CITE <EndNote><Cite><Author>Loman</Author><Year>2014</Year><RecNum>139</RecNum><DisplayText><style face="superscript">21</style></DisplayText><record><rec-number>139</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">139</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Loman, N. J.</author><author>Quinlan, A. R.</author></authors></contributors><auth-address>Institute of Microbiology and Infection, University of Birmingham, Birmingham, UK n.j.loman@bham.ac.uk.
Department of Public Health Sciences, University of Virginia, Charlottesville, VA, USA.</auth-address><titles><title>Poretools: a toolkit for analyzing nanopore sequence data</title><secondary-title>Bioinformatics</secondary-title></titles><edition>2014/08/22</edition><dates><year>2014</year><pub-dates><date>Aug 20</date></pub-dates></dates><isbn>1367-4811 (Electronic)
1367-4803 (Linking)</isbn><accession-num>25143291</accession-num><urls><related-urls><url>, commands `poretools fasta` and `poretools fastq`.These reads were mapped to the de novo assembly of the H125160566 Illumina data using the LAST aligner with the paraments ‘lastal –s 2-T 0 –Q 0 –a 1’ ADDIN EN.CITE <EndNote><Cite><Author>Frith</Author><Year>2010</Year><RecNum>142</RecNum><DisplayText><style face="superscript">16</style></DisplayText><record><rec-number>142</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">142</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Frith, M. C.</author><author>Hamada, M.</author><author>Horton, P.</author></authors></contributors><auth-address>Computational Biology Research Center, Institute for Advanced Industrial Science and Technology, Tokyo 135-0064, Japan. martin@cbrc.jp</auth-address><titles><title>Parameters for accurate genome alignment</title><secondary-title>BMC Bioinformatics</secondary-title><alt-title>BMC bioinformatics</alt-title></titles><pages>80</pages><volume>11</volume><edition>2010/02/11</edition><keywords><keyword>Base Sequence</keyword><keyword>Computational Biology/methods</keyword><keyword>*Genome</keyword><keyword>Molecular Sequence Data</keyword><keyword>Proteins/chemistry</keyword><keyword>RNA/chemistry</keyword><keyword>Sequence Alignment/*methods</keyword><keyword>Sequence Analysis, RNA</keyword></keywords><dates><year>2010</year></dates><isbn>1471-2105 (Electronic)
1471-2105 (Linking)</isbn><accession-num>20144198</accession-num><work-type>Research Support, Non-U.S. Gov't</work-type><urls><related-urls><url>. Miscalled bases (assuming no SNPs and perfect alignment) were determined by parsing the alignment output. If two alignments overlapped by >75%, the alignment with the lower E-value was discarded. To investigate whether some k-mers have a higher occurrence of being missed by the MinION detection, all deletions of length k (where k = 1 to 6) in the MinION reads were extracted from a Samtools pileup (‘samtools mpileup –BQ0 –f <reference.fasta> <sorted.bam>’) file derived from mapping of the MinION reads to the Illumina assembly of the same strain. The distribution of deletion lengths was calculated, as was the frequency of each k-mer (normalised as the number of deletions of a k-mer proportional to the total occurrences of that k-mer) in the H125160566 lllumina assembly. The z-score for each k-mer deletion was calculated, according to z = x – μ / σ where x is the proportion for a particular k-mer, μ is the mean and σ is the standard deviation for the proportion of deletions across all k-mers of that length. A similar process was used to analyse the inserted sequences, with the exception that their frequency was not normalised against the reference genome. Substitutions were identified from the Samtools pileup alignment of MinION reads against the H125160566 Illumina assembly.A hybrid Illumina MinION assembly was generated using SPAdes as above (v3.1.1 ADDIN EN.CITE <EndNote><Cite><Author>Bankevich</Author><Year>2012</Year><RecNum>144</RecNum><DisplayText><style face="superscript">18</style></DisplayText><record><rec-number>144</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">144</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Bankevich, A.</author><author>Nurk, S.</author><author>Antipov, D.</author><author>Gurevich, A. A.</author><author>Dvorkin, M.</author><author>Kulikov, A. S.</author><author>Lesin, V. M.</author><author>Nikolenko, S. I.</author><author>Pham, S.</author><author>Prjibelski, A. D.</author><author>Pyshkin, A. V.</author><author>Sirotkin, A. V.</author><author>Vyahhi, N.</author><author>Tesler, G.</author><author>Alekseyev, M. A.</author><author>Pevzner, P. A.</author></authors></contributors><auth-address>Algorithmic Biology Laboratory, St. Petersburg Academic University, Russian Academy of Sciences, St. Petersburg, Russia.</auth-address><titles><title>SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing</title><secondary-title>J Comput Biol</secondary-title><alt-title>Journal of computational biology : a journal of computational molecular cell biology</alt-title></titles><pages>455-77</pages><volume>19</volume><number>5</number><keywords><keyword>*Algorithms</keyword><keyword>Bacteria/*genetics</keyword><keyword>*Genome, Bacterial</keyword><keyword>Metagenomics/*methods</keyword><keyword>Sequence Analysis, DNA/methods</keyword><keyword>Single-Cell Analysis/*methods</keyword></keywords><dates><year>2012</year><pub-dates><date>May</date></pub-dates></dates><isbn>1557-8666 (Electronic)
1066-5277 (Linking)</isbn><accession-num>22506599</accession-num><urls><related-urls><url> with the ‘—careful’ flag, kmers of 21, 33, 55 and 77) with the concatenated MinION reads for that isolate input under the --PacBio flag as recommended by the authors.Determination of island structure and chromosome insertion siteMinION reads in fasta format were mapped to the Illumina contigs of H125160566 that had matched the pHCM1 and pAKU sequence using LAST. The LAST output, was transformed to BLAST format using the maf-convert.py script bundled with the LAST aligner. The BLAST results were then parsed to obtain all reads that had mapped to at least three of the pHCM1- H125160566 contigs This process is described in more detail in the associated Github repository ADDIN EN.CITE <EndNote><Cite><Author>Ashton</Author><Year>2014</Year><RecNum>156</RecNum><DisplayText><style face="superscript">33</style></DisplayText><record><rec-number>156</rec-number><foreign-keys><key app="EN" db-id="ss5zspp0izpxfme5dxappaxh09rw5zasze5w">156</key></foreign-keys><ref-type name="Web Page">12</ref-type><contributors><authors><author>Ashton, Phil</author></authors></contributors><titles><title>MinION analysis</title></titles><dates><year>2014</year></dates><urls><related-urls><url>. Briefly, the output of the LAST mapping is parsed and read-level and hit-level parameters are produced. The read-level parameters produced are: the number of matches to different contigs; and the read length. The hit-level parameters produced are: the Illumina contig matched to; relative orientation of MinION read and Illumina contig; LAST score; hit length; number of positive matches in the hit; number of indels in the hit; the query (MinION read) start position; the query stop; the subject (Illumina contig) start; and the subject stop. Hits are grouped by MinION read and reads that map to more than one contig are selected. The hits are ordered by the query start, thus making it easy to see the order and orientation of contigs reflected in the MinION reads.Confirmatory PCR Primers used to confirm the structure and point of insertion of the island were designed according to the H125160566 sequence (Supplementary Table 3).PCR was performed in a volume of 40 ?l containing 20 ?l of FideliTaq PCR master mix 2X (Affymetrix, USB), 17?l water, 1?l of 10?M of each primer and 1?l of 100 ng/?l DNA.Amplification was performed in a MWG Gradient Thermal Cycler programmed asfollows: initial denaturation at 95°C for 30 sec and then 29 cycles of denaturation(30 s, 94°C), annealing (1 min, 57°C), and elongation (7 min, 68°C), with a final elongation step (7 min, 68°C). The amplified product was electrophoresed at 100V on a 1.6% agarose gel (type II medium electrophoresis grade) in a 0.5 x TBE buffer (45 mM Tris, 45 mM boric acid, 10 mM EDTA [pH 8]). Following electrophoresis the gel was stained in ethidium bromide (1 ?g/ml) and photographed under UV light.Sequencing of PCR ampliconsPCR amplicons were purified by ExoSAP-IT cleanup system according to the manufacturer’s instructions (Affymetrix/USB). DNA sequencing of these amplicons was performed using the same PCR primers by the chain-terminating method of Sanger et al. (1997) at the Genomic Service Unit, Public Health England. Analysis was performed using BLAST programs and database services produced by National Center for Biotechnology Information, Bethesda, MD (ncbi.nlm.).Figure legendsFigure 1: The z-score distribution of k-mers between 3 and 6 bp binned by k-mer GC content.Figure 2: Genetic organisation of the S. Typhi chromosomal resistance island.Legend: Gene names were assigned using BLAST analysis and manual annotation.Figure 3: Comparison of the S. Typhi chromosomal resistance island with two closely related plasmids.Legend: The S. Typhi chromosomal resistance island was compared to the S. Typhi multi-drug resistance plasmid pHCM1 and the plasmid with the highest BLASTN similarity to the island, pRSB107, from an uncultured organism in sewage. Top: pHCM1; middle: genomic island; bottom: pRSB107.Overview to show full length sequence of pHCM1 and pRSB107Zoomed in view to show detail of regions of similarity. Red indicates 100% similarity and blue indicates 99% similarity and the cut-off minimum was set at 800. ................
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