DNA extraction is performed using the QIAamp PowerFecal Pro DNA Kit and the V4-V5 region of 16S rRNA- genes are PCR amplified using barcoded dual-index primers. Primer barcodes and sequences will be provided upon request. Illumina compatible libraries are generated using the Qiagen QIASeq 1-step amplicon kit  and sequencing is performed on the Illumina MiSeq platform in the MMF using a 2×250 Paired End reads, generating 5,000-10,000 reads per sample.  Raw V4-V5 16S rRNA gene sequence data is demultiplexed and processed through the dada2 pipeline into Amplicon Sequence Variants (ASVs). ASVs are identified with the Bayesian RDP classifier up to the genus level and are BLASTed against RefSeq for species-level identification with corresponding alignment statistics. Raw sequence data, ASV abundance and Taxonomy tables (derived from both RDP and BLAST), in addition to bar plots, are generated.  

Included in service is DNA extraction, Illumina library preparation, and Illumina sequencing, as well as the following basic data analysis: pipeline running, ASV count table generation, taxonomy identification, and bacteria abundance bar plots.

DNA extraction from bacterial cultures is performed using the QIAamp PowerFecal Pro DNA Kit. Illumina compatible libraries are generated using the QIAseq FX Library Kit and sequencing runs are performed on the Illumina NextSeq2000 platform in the MMF. NextSeq runs are performed on 2×150 Paired End reads cassettes to generate between 3 to 5 million reads per isolate. SPADES and prokka are used for assembly and annotation of bacterial whole-genomes.

Included in service is DNA extraction, Illumina library preparation, Illumina sequencing and genome assembly and annotation. The following basic data analysis is also included: pipeline running, assembling contigs, gene annotation, and bacteria identification.

DNA extraction is performed using the QIAamp PowerFecal Pro DNA Kit and Illumina compatible libraries are generated using the QIAseq FX Library Kit. Sequencing runs are performed on the Illumina NextSeq 2000 platform in the MMF using the 2×150 Paired End reads cassette. Shotgun samples are taxonomically profiled using Kraken2 while deep shotgun sequencing can be assembled into metagenomic contigs using metaSPADES and annotated using prokka upon request. Metagenomic contigs may also be taxonomically identified using Kraken2. We can also help quantify abundances of genes of interest using diamond, bowtie2, and/or shortBRED. We recognize that there is no ‘one-size-fits-all’ analysis approach for shotgun metagenomics and are open to tailoring analyses to better fit with the goals of your project. We offer Shallow and Deep shotgun sequencing services.

Shallow shotgun sequencing to generates greater than 10 million reads per sample.

Deep shotgun sequencing to generate greater than 50 million reads per sample.

Both services include DNA extraction, Illumina library preparation, and Illumina sequencing. Also included is basic data analysis: pipeline running, assembling contigs, functional annotation, and bacteria abundance bar plot.

Metabarcoding (followed by end-point PCR) of specific hypervariable regions within the 16S rRNA gene enables estimation of the relative abundance of microbes in a sample. 16S amplicon sequencing using PCR provides qualitative information on the different taxa present within a community.  The above method can be coupled with quantitative PCR (qPCR) to determine total bacterial load.  Quantitative PCR measures the number of copies of the 16S V4-V5 region and quantifies bacterial load within each sample. This method utilizes degenerate primers and hence predicts only relative abundance and does not account for 16S copy number variations among different bacterial species. Copy numbers will be normalized per sample if mass is provided.

RNA extraction from bacterial cultures is performed using Maxwell RSC simplyRNA Blood kit on Maxwell RSC. For fecal samples, we use the RNeasy PowerFecal Pro Kit. Illumina compatible libraries are generated using NEBNext® Ultra™ II Directional RNA Library Prep Kit. The MMF performs ribosomal RNA depletion using NEBNext rRNA Depletion Kit (bacteria) and NEBNext Globin and rRNA Depletion Kit (human/mouse/rat) prior to library preparation.  Libraries are sequenced using 2x100bp reads on NextSeq 2000.  Samples are aligned to supplied reference genome, and reads counts are binned by genes using associated annotations. Differential gene expression analysis and pairwise comparisons are performed using DESeq2.  The MMF provides a list of significantly (default FDR 5%) differentially expressed genes compared between requested conditions.