A Comprehensive Workflow for Microbial Genome Sequencing Results

A Comprehensive Workflow for Microbial Genome Sequencing Results Bench Work objective of the present study was to ign, test, troubleshoot, and publish a mprehensive workflow for taking a earcher from a swab to a microbial ome publication; enabling even a lab h limited resources and bioinformatics erience to perform it. UC Davis Genome Center 1 University of California, Davis; 2 ithree Institute, University of Sydney, Australia ume a starting point of wanting to isolate an organism from cular environment and needing to identify it. Users starting known organism should proceed to "Library Preparation and ncing”. e cover the steps necessary to take a sample through plating, n streaking, overnight growth, creating a glycerol stock, 16 s d preparation for Sanger sequencing. Sanger Sequence Processing section we identify multiple software programs that allow earcher to view and edit the genetic sequence. We detail the ages and disadvantages of particular programs and explain o quality trim the reads, reverse complement and align the and generate a consensus sequence. This is easiest to do y via a chromatogram allowing the user to see the trace and s the sequences manually. me assembly typically consists of data cleaning (quality Assembly oice of sequencing technology and of library preparation d for genome sequencing is ever-changing and much-debated. e recommend using Illumina Mi. Seq for reasons of cost, of coverage, and length of reads. Furthermore, the assembly e, A 5 -miseq, requires Illumina data and is optimized for the reads from the Mi. Seq. Library Preparation and Sequencing ssroom or undergraduate research setting the researchers ot have a particular bacterial species in mind. In this case it is ary to screen the 16 S Sanger sequencing results for possible e project candidates. We recommend starting with the results, then continuing onto the Genomes Online Database , and simply Google searching. In many cases it will be to first build a phylogenetic tree to aid in identification since S results may not be Identify/Choose Microbe Madison I. Identify/Choose Microbe • • • Swab Plate Dilution Streak (X 2) Overnight Culture DNA Extraction 16 s PCR • Interpretation of A 5 stats • Verification of 16 S Sequence • Phylosift • Seq. Trace • Geneious • Custom Script • Download and Install A 5 • Running A 5 • • BLAST 16 S Interpret the Results GOLD Align the 16 S Sequences using Align Sequences Nucleotide BLAST • Library Preparation • Kit Options • Considerations in Library Preparation • Multiplexing Gen. Bank Submission Making a Phylogenetic Tree Annotation Library Preparation and Sequencing Assembly Verification of the Assembly Aaron E. Guillaume Jenna M. David A. 1 Coil , 1 Dunitz , Sanger Sequence Processing Bench Work Jonathan A. 1 Eisen 2 Darling , 1 Jospin , 1 Lang , Introduction From Swab to Publication The sequencing and de novo assembly of microbial genomes has already yielded enormous scientific insight revolutionizing a diverse collection of fields, from epidemiology to ecology. In the past two decades increasing advances in DNA sequencing technology have led to the creation of a wide variety of options for DNA library preparation, sequencing and assembly. Each option comes with its own advantages and disadvantages in terms of complexity, expense, computing power, time, and experience required. This workflow was designed in an attempt to democratize the process of microbial sequencing and de novo assembly, in order to make them accessible to low funded labs or even classrooms on a massive scale. VI. Verificatio There are three portions to th The first is the overall "quality examining the stats provided contig N 50). The second is ve sequenced is the organism of sequence with BLAST. The thi genome, which is difficult to m reference (representative exa available. VII. Annotation There a number of differe annotation of bacterial genom PGAP and others. Genomic an coding regions and attemptin and their biological function. VIII. Making a P There are two points during th phylogenetic tree may be use candidate organisms by Sange assembly of the genome. The the additional length and imp 16 S sequence may generate a identification of the candidate single species clade), for nami clade containing only membe of the species are not found o of the organism into a phyloge The outline of this step, is to u (RDP) to generate an alignmen and an out-group, following b building with Fast. Tree and vie Dendroscope. IX. Gen. Bank S This section describes how to applicable) as a Whole Genom Embank. We also recommend genome themselves, since sub can be prohibitively complicat shared with the DNA Data Ban addition, genomes from Gen. B and are annotated there as w What Do Fill out a post