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AAT Bioquest

What is the basic process of short-read sequencing ?

Posted June 4, 2024


Answer

Although there are a variety of short-read sequencing platforms, the basic process of short read sequencing is similar in all. It involves 7 main steps: 

  • Nucleic acid isolation: A kit or automated DNA extractor is used to isolate high quality DNA or RNA
  • Fragmentation and library preparation: The genomic DNA is fragmented and the fragments of interest are enriched by hybridization probe capture or PCR. Downstream processing and identification of samples is enabled by the addition of small artificial DNA sequences or adapters. 
  • Massively parallel sequencing: Reads between 50 and 300 bases long are generated after simultaneously reading the sequence of bases in several different DNA fragments
  • Quality control: Qualified reads are collected after checking the read data and performing read quality assessment to ensure they are worth collecting for analysis. 
  • Read alignments: Each short read is aligned to the specific place in the genome it represents. This is done using a reference genome sequence with specialist software. 
  • Variant calling: Variations in sequences between the reference sequence and the sample DNA are identified and noted. 
  • Variant annotation: Software is used to annotate variations and predict the likely effect that a DNA variant will have on a protein and/or a phenotype.  

 

Additional resources

Cost-conscious generation of multiplexed short-read DNA libraries for whole-genome sequencing

Next Generation Sequencing (NGS)

MagaDye™ 535-ddGTP