A Single Tube Long Fragment Read(stLFR) Sequencing Technology Based On Co-Barcode Method Research

To date the vast majority of individual whole genome sequences lack information regarding the order of single to multi-base variants transmitted as contiguous blocks on homologous chromosomes.In addition,most of the species that have been sequenced have missing sequence information in their reference genome sequences,such as large structural variations,repetitive sequences and other regions that are difficult to analyze with current technologies.Therefore,a simple,fast and economical haplotyping technology is needed.The technology should not rely on automated liquid separation equipment,complex experimental operations and a large amount of time investment.So long fragment sequencing technology based on single tube,virtual physical separation and joint tag is the best choiceThis research developed a low-cost single-tube-long fragment sequencing technology(stLFR)based on co-Barcoding.This technology,combined with the second-generation sequencing platform,can indirectly obtain the average reading length of 50kb.At the same time,it has the characteristics of high accuracy and low cost of second-generation sequencing technology.50million beads can capture more than 8million genomic DNA molecules with a length of 20-300kb,and Every long DNA molecule can be covered about 20%.High-quality mutation detection results were obtained by using stLFR to analyze the human genome of NA12878.In addition,stLFR technology could also gain phasing Contig N50 up to 15Mb.stLFR technology can detect complex structural variations.Meanwhile,the de novo assembling contig N50 of stLFR is about 100kb and the scaffold N50 is 30Mb.The total cost of stLFR is about 10,000 yuan to obtain 30X coverage of human genome.The cost of a traditional WGS is about 8,000 yuan.Pacbio,the most mature of the 3rd generation sequencing technology,costs 20 times more than WGS.The research results show that stLFR is a low cost and high accuracy sequencing technology that can perform haploid Phasing detection,SV detection,or de-novo genome assembly.