150 bp Next Generation Biome Sequencing

by | Oct 7, 2024 | Uncategorized

To perform Next Generation Sequencing (NGS) of soil bacteria genomes at the 150 bp read length level, specifically using platforms like Illumina (which is the most common for short-read sequencing like 150 bp), you need a combination of lab equipment, consumables, reagents, and bioinformatics tools. Here is a comprehensive list divided into categories:

1. Sample Collection and Preparation

  • Sterile Soil Collection Containers: To collect and store soil samples in a contamination-free environment.
  • Sterile Gloves: To avoid contamination during soil sample collection and handling.
  • Sterile Spatulas or Scoops: For transferring soil samples.
  • DNA Extraction Kits: To extract bacterial DNA from soil. Examples include:
  • DNeasy PowerSoil Kit (QIAGEN): Specifically designed for extracting microbial DNA from soil.
  • ZymoBIOMICS DNA Miniprep Kit (Zymo Research): Efficient for low-biomass and challenging soil samples.
  • Microcentrifuge Tubes (1.5 mL): For handling and processing extracted DNA.
  • Centrifuge: For pelletizing soil material and separating bacterial cells during DNA extraction.
  • Vortex Mixer: To ensure proper mixing during DNA extraction protocols.

2. DNA Quantification and Quality Control

  • Nanodrop Spectrophotometer: To measure DNA concentration and assess purity (260/280 and 260/230 ratios).
  • Qubit Fluorometer: For accurate DNA quantification using fluorescence-based assays (especially important for low concentrations).
  • Agarose Gel Electrophoresis System: For assessing the size and integrity of the DNA.
  • Agarose Gel Powder
  • Gel Casting Tray and Comb
  • Gel Red or SYBR Safe Stain: For visualizing DNA under UV or blue light.
  • Gel Electrophoresis Running Buffer (e.g., TAE or TBE)
  • UV Transilluminator or Blue Light Gel Imager

3. Library Preparation

  • NGS Library Preparation Kit: A kit that provides all reagents to prepare DNA libraries for sequencing, such as:
  • Illumina DNA Prep Kit: Commonly used for preparing DNA for short-read sequencing.
  • Nextera XT DNA Library Prep Kit (Illumina): Suitable for small genomes like bacterial genomes.
  • Fragmentation Enzymes: If mechanical shearing is not used, enzymatic fragmentation kits like NEBNext Ultra II FS DNA Library Prep can be used to fragment DNA to 150 bp.
  • Magnetic Beads (AMPure XP): For DNA purification during library preparation steps.
  • PCR Thermocycler: For amplification and adapter ligation during library preparation.
  • DNA Clean-Up and Size Selection Kits: For purifying and selecting fragments around 150 bp. AMPure XP beads are commonly used for this step.
  • Index Adapters: To label each sample with unique barcodes, allowing multiple samples to be sequenced in one run (e.g., Nextera Index Adapters).

4. Sequencing Platform

  • Illumina Sequencing Instrument: Depending on your throughput and read-length requirement (150 bp), popular models include:
  • Illumina MiSeq: Ideal for smaller projects or when 2 x 150 bp read length is sufficient.
  • Illumina NextSeq: Suitable for medium to large projects with more throughput.
  • Illumina NovaSeq: Used for very large projects requiring massive throughput.
  • Sequencing Flow Cells: Specific to the sequencing platform you’re using (e.g., MiSeq Flow Cell).
  • Sequencing Reagents Kit: Contains all the necessary reagents (buffers, enzymes) for sequencing.
  • MiSeq Reagent Kit v2 (300 Cycles): For 150 bp paired-end sequencing (150 bp read length × 2 = 300 total cycles).

5. Supporting Equipment and Consumables

  • Magnetic Rack: Used for bead-based cleanups during DNA extraction or library preparation.
  • Multichannel Pipettes and Pipette Tips: For accurate liquid handling during library preparation and sequencing.
  • Plate Seals and PCR Plates: For PCR during library preparation and barcoding.
  • Freezer (-20°C and -80°C): To store reagents and samples.
  • Refrigerator (4°C): For temporary reagent storage.
  • Sterile Water or Nuclease-Free Water: For dilutions and reactions.
  • Ethanol (70% and 100%): For DNA purification steps.
  • TE Buffer or Elution Buffer: For eluting purified DNA.

6. Bioinformatics Tools

Once sequencing is complete, bioinformatics tools are essential for processing and analyzing the data:

  • BaseSpace (Illumina): Cloud-based bioinformatics platform for analyzing data generated on Illumina machines.
  • FASTQC: For quality control checks on raw sequencing reads.
  • Trimmomatic or Cutadapt: To trim adapters and low-quality bases from raw reads.
  • SPAdes or Velvet: For de novo genome assembly of bacterial genomes.
  • Bowtie2 or BWA: For aligning reads to a reference genome if applicable.
  • MEGAHIT or MetaSPAdes: For metagenomic assembly of soil bacterial communities.
  • Kraken2, MetaPhlAn, or QIIME2: For taxonomic classification and microbial community profiling.

7. Optional and Specialized Equipment

  • Sonicator or Covaris System: For mechanical DNA fragmentation, if enzymatic fragmentation is not preferred.
  • Automated Liquid Handling System: To improve accuracy and throughput during library prep (e.g., Beckman Coulter Biomek or Hamilton Microlab).
  • Bioanalyzer or TapeStation (Agilent): For high-precision sizing and quantification of DNA fragments, libraries, or PCR products.

Summary Workflow

  1. Soil Collection & DNA Extraction → Collect soil, extract bacterial DNA.
  2. DNA Quality Check → Use Nanodrop, Qubit, and Gel Electrophoresis to quantify and assess DNA quality.
  3. Library Preparation → Fragment DNA, add adapters/barcodes, amplify DNA.
  4. Sequencing → Load libraries into an Illumina sequencer for 150 bp paired-end sequencing.
  5. Bioinformatics → Process raw data, align reads, assemble genomes, or profile bacterial communities.

Let me know if you need more details about any specific step!