AOAC-RI ERP Book Micro Jan 19 2017

OMAMAN-35 A : Collaborative Study Manuscript For ERP Use Only January 2017

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 9 dsDNA Qubit fluorometry and purity was assessed using a NanoDrop N-1000 spectrophotometer; 10 see Table 7 for quantification and quality control results. 11 2. Library Preparation for Sequencing 12 The sequencing libraries were prepared according to the QiaseqFX DNA Library kit protocol. 13 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. 16 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 18 spiked at 5% (vol/vol) of the library pool, as recommended for low-diversity libraries. The 19 pooled libraries were loaded and sequenced using a MiSeq V2 500-cycle reagent kit with 250bp 20 paired-end reads. 21 3. Data Handling and Analysis 22 Raw data sequences, in the form of paired FASTQ files, were first analyzed using the FastQC 23 software (V0.11.5) to determine quality. Sequence reads were then submitted to a scripted 24 pipeline, A5-Miseq (v20160825), which automates the data cleaning, alignment, and assembly. 25 Finally RNAmmer (v1.2, server) was used to extract the 16S rRNA sequence from the draft 26 assemblies. The isolated 16S sequence was provide in the form of FASTA files. 27 References 28 A5-miseq: an updated pipeline to assemble microbial genomes from Illumina MiSeq data. Coil D, Jospin 29 G, Darling AE. Bioinformatics. 2015 15;31(4):587-9. Doi: 10.1093 30 Results 31 1. 16S rRNA SSU sequences. Provided in Appendix A. In addition to sequences provided in 32 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. 34 Table 7. Isolate gDNA Quality Check Data 35 Isolate ID ng/µL (Qubit) A260/A280 (nanodrop) A260/230 (Nanodrop) AOAC Research Institute Expert Review Panel Use Only

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