Experimental Workflow



   The samples were prepared according to the Illumina TruSeq Nano DNA library preparation guide or TruSeq DNA PCR-free library preparation guide. The libraries were sequenced using Illumina platform.



◦ Experimental Procedure


  • DNA fragmentation
Each sequenced sample is prepared according to the Illumina TruSeq DNA sample preparation guide to obtain a final library of 300-400 bp average insert size. One microgram (TruSeq DNA PCR-free library) or 100 nanogram (TruSeq Nano DNA library) of genomic DNA is fragmented by covaris systems, which generates dsDNA fragments with 3' or 5' overhangs.

  • End repair and size selection
The double-strand DNA fragments with 3' or 5' overhangs are converted into blunt ends using an End Repair Mix. The 3' to 5' exonuclease removes the 3' overhangs, and the polymerase fills in the 5' overhangs. Following the end repair, the appropriate library size is selected using different ratios of the Sample Purification Beads.

  • Adenylation of 3' end
A single 'A' nucleotide is added to the 3' ends of the blunted fragments to prevent them from ligating to one another during the adapter ligation reaction. A corresponding single 'T' nucleotide on the 3' end of the adapter provides a complementary overhang for ligating the adapter to the fragment.

  • Adapters ligation
Multiple indexing adapters are ligated to the ends of the DNA fragments to prepare them for hybridization onto a flow cell.

  • DNA fragments enrichment (TruSeq Nano DNA library only)
PCR is used to amplify the enriched DNA library for sequencing. The PCR is performed with a PCR primer solution that anneals to the ends of each adapters.

  • Library validation
Macrogen performs quality control analysis on the sample library and quantification of the DNA library templates.

   For cluster generation, the library is loaded into a flow cell where fragments are captured on a lawn of surface-bound oligos complementary to the library adapters. Each fragment is then amplified into distinct, clonal clusters through bridge amplification. When cluster generation is complete, the templates are ready for sequencing. Illumina SBS technology utilizes a proprietary reversible terminator-based method that detects single bases as they are incorporated into DNA template strands. As all 4 reversible, terminator-bound dNTPs are persent during each sequencing cycle, natural competition minimizes incorporation bias and greatly reduces raw error rates compared to other technologies. The result is highly accurate base-by-base sequencing that virtually eliminates sequence-context-specific errors, even within repetitive sequence regions and homopolymers.

   The Illumina Platform generates raw images and base calling through an integrated primary analysis software called RTA (real time analysis). The BCL/cBCL (base calls) binary is converted into FASTQ using illumina package bcl2fastq2-v2.20.0. The demultiplexing option (--barcode-mismatches) was set to perfect match (value : 0).