Microbial Sequencing & Analytics Core

• Using tissue samples, bodily fluids, FFPE slices, etc. MSAAR will isolate & purify the appropriate nucleic acid (DNA or RNA).
• First, to remove the genetic material from the cells, cell lysis must occur using mechanical and/or enzymatic measures, followed by the removal of proteins and other cellular debris and finally, purification of the DNA or RNA by removing any remaining contaminants.
• Once the nucleic acids have been isolated and purified. We perform necessary quality control measures to check for
  • nucleic acid purity
  • sample concentration [ng/uL]
  • integrity of the gDNA or Total RNA extracted

All samples in the MSAAR facility undergo quality control (QC) procedures, ensuring the quantity and the quality of the nucleic acids and prepared libraries are fit to move forward in preparation for NGS (Next Generation Sequencing). Our QC services are also available to any investigator within the Cleveland Clinic and its affiliates.

Using an illumina sequencing platform, the core houses two illumina sequencing instruments

• MiSeq – debut as one of illumina's first benchtop sequencers. This instrument can accommodate long 600 bp (i.e. 2 x 300 bp) reads lengths
  • Data output ranges:
    • 540 Mb – 15 Gb
    • 1 Million (M) – 25 Million (M) Single Reads
    • 2 M – 50 M Paired-end reads
  • Max Read length: 2x 300 bp
• MiSeq i100 Plus – created as the new standard for NGS featuring the fastest run times using proprietary XLEAP-SBS chemistry & integrated data analysis
  • Data Output ranges:
    • 1.5 Gb – 30 Gb
    • 5 Million (M) – 100 Million single reads
    • 10 M – 200 M paired end reads 
  • Max read Lenth: 2 x 300 bp

• Amplicon Sequencing Analysis (16S rRNA, 18S rRNA, ITS)
• OTU clustering based e.g. Qiime and Mothur
• Sequence variant (SV) based e.g. DADA2 and Deblur
• 16s RNA amplicon sequencing based functional analysis (PiCRUSt and FAPROTAX)
• Shotgun sequencing based metagenomics, metatranscriptomics and RNA-Seq analysis
• Quality control and filtering (e.g. Quality based trimming, adapter/primer sequence removal, host sequence removal)
• Individual read based taxonomic and functional characterization (e.g. HUMAN2, Kraken and Metaphlan2)
• Individual read based antibiotic resistance gene analysis
• Sequence variant analysis
• De-novo genome, metagenome, metatranscriptomics assembly (e.g. SPADES, MetaSPADES, IDBA, Meta-IDBA)
• Assembly validation, gene calling and annotations (KEGG, CAZY, COGG etc.)
• De-novo genome reconstruction from metagenome assembly (i.e. contigs)
• Strain level characterization and comparative genomics of the reconstructed genomes
• Antibiotics resistance gene analysis using assembly (i.e. contigs)

• Power Calculations (sample size estimation)
• Differential feature selection analysis
• Confounding factor identification and correction
• Biomarker identification
• Association analysis (e.g. MGWAS, WGCNA and GLM’s)
• Machine learning based model creation and validation (prediction and classification)
• Publication quality plot generation and sequence data submission (e.g. EMBL and NCBI)

• Microbial species/strain selection for Metabolic modeling
• Constraint based metabolic model creation of microbial genomes
• Constraint based metabolic model creation from host specific RNA-Seq or microbiome data
• Creation of host-microbiome metabolic model from microbial and host-specific metabolic models
• Simulation and flux balance analysis on microbial community model and/or host-microbiome metabolic model
• Identification of metabolites that can help increase and/or decrease the abundance of microbial and host cells
• Designing in-vivo and/or in-vitro experiments to validate metabolic modelling results

• What is library prep?
  • Library prep is the process of converting gDNA or cDNA into a library of fragments that are uniformly sized, containing the appropriate oligonucleotide adapters needed for NexGen sequencing to occur
• What types of library preps does MSAAR offer?
  • Amplicon based library prep – (16S used for bacterial studies & ITS is used for fungal studies)
    • Amplification of the highly variable v4 region of the 16S rRNA gene is commonly used for:
      • Investigation in bacterial phylogeny and taxonomic comparisons
      • Suited for the identification or comparison of bacteria or fungi present within a given sample
      • Can help differentiate between closely related microbial species
    • Amplification of other variable regions of the 16S rRNA gene can be performed as needed
  • Nextera XT (insert size varies) or illumina DNA Prep(target insert size 350bp)
    • Suited for:
      • completing the genomes of known microbes,
      • mapping genomes of novel microbial organisms, or
      • comparing genomes across several different samples
      • evaluate bacterial diversity,
      • generating reference genomes
  • Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit
    • Library prep targeting encoding and non-encoding sequences
    • Suited for studies of gene expression within microbial community
    • Along with identifying which microbes are present this also provides insight of microbial function

For more information regarding project design & bioinformatical analysis of NGS data – contact:

Naseer Sangwan Ph.D. | MSAAR Core Director | Associate Staff | [email protected]