Nanopore Sequencer Precision Verification And Application

In recent years,with the development and popularization of next-generation sequencing technology,nanopore sequencing technology has gradually entered people's field of vision.Because of its advantages such as long sequencing reads,short sequencing cycles,and simple operations,it has become the target of many research staff.Therefore,the development of nanopore sequencers with longer sequencing reads,shorter sequencing cycles,and more convenient operations has become an urgent problem for developers of nanopore sequencers,but under the premise of pursuit of longer sequencing reads,shorter sequencing cycles,simpler operation and other performance,how to ensure the precision of the nanopore sequencer has become an urgent problem.This article aims to verify the precision of the nanopore sequencer and prepare nanopore sequencing libraries of different species to testify the application and performance of nanopore sequencer.The results is as follows:Vector plasmid digestion and artificial synthesis of test library fragments are performed to prepare linearized vectors and test library fragments.Linearized vectors and test library fragments are cloned in one step to prepare the preparation plasmids for double-stranded test library fragments and the preparation plasmids for single-stranded test library fragments.The preparation plasmids for double-stranded test library fragments was used as a template and amplified by PCR.BsaI restriction endonuclease sites and different sticky ends were added to both ends of the double-stranded test library fragment.The purification product of PCR amplification reaction was digested by BsaI restriction enzyme and purified to prepare sufficient double-stranded test library fragments to be ligated.Designing sequencing adapter,sequencing adapter and double-stranded test library fragments to be ligated are ligated,and the ligation product is recovered by gel to prepare double-stranded test library,which is experimental material for sequencer accuracy verification.The single-stranded test library fragment preparation plasmid was transformed into E.coli JM109,which was subjected to night culture,isolation culture supernatant,and target M13 phage single-stranded genome extraction to obtain the target M13 phage single-stranded genome containing the single-stranded test library fragment.The target M13 phage single-stranded genome is annealed with a complementary fragment,then digested by EcoRI and XbaI restriction enzymes and purified by magnetic beads to prepare single-stranded test library fragment to be annealed.Designing sequencing adapter,sequencing adapter and single-stranded test library fragment to be annealed are annealed together to prepare single-stranded test library,which is experimental material for sequencer accuracy verification.Use a nanopore sequencer to sequence the prepared double-stranded test library and single-stranded test library,and compare the nanopore sequencing results of the double-stranded test library and single-stranded test library with the nucleic acid sequence information of the double-stranded test library and single-stranded test library.The accuracy of the nanopore sequencer for sequencing double-stranded test libraries and single-stranded test libraries is obtained.The accuracy of the nanppore sequencer for sequencing double-stranded test library fragments is 95.0%,and the accuracy of the nanopore sequencer for sequencing single-stranded test library fragment is 86.3%.On the basis of verifying the precision of the nanopore sequencer,the human and mouse SCFV-Fc light chain genes were sequenced and analyzed using the nanopore sequencer,and the practical application value of the nanopore sequencer was explored.The following results were obtained: The 5 ends of the SCFV-Fc light chain gene were digested with EcoRI restriction enzymes,and the 3 ends were digested with HindIII restriction enzyme,and the purified product was constructed.The sequencing results of the human and mouse SCFV-Fc light chain gene digested libraries were compared with the sequence information of human and mouse SCFV-Fc light chain gene digested libraries,and the accuracy of the nanopore sequencer in practical applications is shown: the accuracy of the nanopore sequencer for for human SCFV-Fc light chain gene digested library fragments is 95.4%,and the accuracy of the nanopore sequencer for mouse SCFV-Fc light chain gene digested library fragments is 95.6%.Based on the above results,there is still a certain error rate in nanopore sequencing,and nanopore sequencing instrument needs to be further improved.