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3M Molecular Detection Assay 2 –
E. coli
O157 (including H7) collaborative isolates needing additional
1
identification by 16S rRNA sequencing.
2
Data generated by: NSF International,789 North Dixboro, Ann Arbor, MI 48105
3
Methodology
4
1.
DNA Extraction for Sequencing
5
Sterile loops were used to collect sufficient mass of isolated colonies. Colonies were suspended
6
in solution of 180 µL of Qiagen ATL buffer and 20 µL proteinase K. Samples were incubated
7
overnight at 56 ± 1°C to complete lysis. The sample lysate was then purified via Qiagen silica-
8
gel spin-columns and eluted with 10 mMTris HCL. The gDNA products were quantified via
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dsDNA Qubit fluorometry and purity was assessed using a NanoDrop N-1000 spectrophotometer;
10
see Table 7 for quantification and quality control results.
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2.
Library Preparation for Sequencing
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The sequencing libraries were prepared according to the QiaseqFX DNA Library kit protocol.
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Tris HCL buffer was used as the diluent for the input gDNA. A manual hand-pump centrifuge
14
(RPI HandyFuge) was used for all 96-well plate centrifuge steps. A Bio-Analyzer was not
15
available for the optional QC product cleanup.
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Sample libraries were quantified via fluorometry, normalized, and pooled at 10pM according to
17
the MiSeq V2 Reagent kit protocol. Illumina PhiX V3 Control DNA was diluted to 12.5 pM and
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spiked at 5% (vol/vol) of the library pool, as recommended for low-diversity libraries. The
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pooled libraries were loaded and sequenced using a MiSeq V2 500-cycle reagent kit with 250bp
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paired-end reads.
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3.
Data Handling and Analysis
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Raw data sequences, in the form of paired FASTQ files, were first analyzed using the FastQC
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software (V0.11.5) to determine quality. Sequence reads were then submitted to a scripted
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pipeline, A5-Miseq (v20160825), which automates the data cleaning, alignment, and assembly.
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Finally RNAmmer (v1.2, server) was used to extract the 16S rRNA sequence from the draft
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assemblies. The isolated 16S sequence was provide in the form of FASTA files.
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References
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A5-miseq: an updated pipeline to assemble microbial genomes from Illumina MiSeq data.
Coil D, Jospin
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G, Darling AE. Bioinformatics. 2015 15;31(4):587-9. Doi: 10.1093
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Results
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1.
16S rRNA SSU sequences. Provided in Appendix A. In addition to sequences provided in
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Appendix A, FASTA files are also being provided along with this report. However, due to the
33
insecure nature of these FASTA files, they are being provided only as a courtesy.
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Table 7. Isolate gDNA Quality Check Data
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Isolate ID
ng/µL (Qubit)
A260/A280 (nanodrop)
A260/230 (Nanodrop)
OMAMAN-35 A : Collaborative Study Manuscript
For ERP Use Only
January 2017
AOAC Research Institute
Expert Review Panel Use Only