Next Generation Sequencing (NGS) Illumina Library Prep

We perform Illumina NGS library preparation from a variety of starting materials, including genomic DNA, PCR products, total RNA, and mRNA. These libraries can be sequenced on various Illumina platforms, such as the MiSeq and MiSeq i100 Plus Personal Systems, NextSeq 2000, NovaSeq X, and others. Applications include de novo sequencing, whole-genome and targeted DNA resequencing, sequencing of individual chloroplast genomes, metagenomic analysis of environmental samples, RNA profiling, and more.

For a typical shotgun genome sequencing project provide ~1.0 ug of double-stranded DNA, with the minimal concentration of 50 ng/ul in H2O or EB (Qiagen) buffer, OD (260/280)>1.8. Please provide a photo of an agarose gel documenting the DNA quality. For all other types of samples and additional information please contact Alina Akhunova at akhunova@ksu.edu

NGS Workflow

1) Sample Quality Controls available at the IGF:

  • NanoDrop-based OD260/280 and OD260/230 measurements
  • Qubit-based quantification
  • PicoGreen-based quantification using the Synergy H1 Hybrid Multi-Mode Microplate Reader
  • total RNA/mRNA integrity assessment with the Agilent 2100 Bioanalyzer
  • genomic DNA quality assessment with the Agilent 4150 Tape Station
  • PCR products quality control with the Agilent 2100 Bioanalyzer or 4150 TapeStation.

2) NGS libraries: Depending on the number of submitted samples, libraries can be constructed using the Biomek FXP Laboratory Automation Workstation. This system handles all aspects of liquid handling, including pipetting, dilution, dispensing, and integration.

3) Library size selection: We use both bead-based and Blue Pippin-based size selection.

4) NGS Library QC:

  • Qubit-based quantification
  • quality assessment with the Agilent 2100 Bioanalyzer or 4150 Tape Station
  • qPCR-based quantification using Roche/KAPA reagents

5) Library dilution, pooling, sequencing or shipping to a NovaSeq facility.

6) We use Illumina BaseSpace to share sequencing results. If you do not have an account, please create one and select the free account option—this is sufficient to access your data. Once your account is set up, please let us know which email address you used.

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