Whole Genome Sequencing

BRIEF DESCRIPTION & SCOPE OF WORK:

Sample requirement:

  •  gDNA: Submit 2-4 µg of RNA-free genomic DNA (NGS grade) of molecular weight           > 40kb, Nanodrop A260/280 ratio >1.8; A260/230=2.0- 2.2; and at a concentration of ~50 ng/µl (dissolve in low TE buffer).

Sample Quality Control:

  • NGS grade High quality DNA will be isolated from the supplied sample using commercially available kits.
  • NGS grade plasmid isolation by using commercially available kits.
  • The qualification and quantification of gDNA will be carried out by denatured agarose gel electrophoresis and NanoDrop/Qubit Fluorometer analysis.

Library Preparation:

  • DNA Seq library will be prepared by using Tru Seq DNA Sample Prep Kit. Library validation will be carried out using High Sensitivity D1000 Screen tape using Agilent TapeStation.

Sequencing: 

  • Sequencing of library on NextSeq v2.0 Chemistry and with 2 x150 bp paired-end read module
  • Delivery of approx. 1 GB data per sample.

Bioinformatics Analysis: 

  • Quality filtration of reads,
  • Mapping of high quality reads to the reference genome
  • Alignment summary
  • Consensus sequence
  • Gene identification using gtf/gff,
  • SNP/Indels identification,
  • SNP/Indels annotation,
  • Compiled report

 

Delivery Time :

  • Provided that there are no unexpected difficulties (biological or technical), 5-6 weeks from the date of QC qualified samples received. All samples to be provided in a single lot.

 

Working Flow Chart:-

  • DNA QC with agarose gel,
  • Nanodrop, quantification with Qubit fluorometer,
  • Library preparation with TruSeq sample prep kit,
  • Library QC on Agilent Tape station,
  • Sequencing on Illumina NextSeq 600 platform with 2X150 bp PE chemistry,
  • Generation of 1 to 1.5 Gb data per sample.
  • Reference based Bioinformatics Analysis