Studies On Y Chromosome Hypervariable Regions And Mitochondrial Genome For Distinguishing Monozygotic Twins

Objective: Monozygotic twins(MZT)discrimination has always been a challenge in Forensic Genetics.Since MZT are from one fertilized egg,they share almost the same genetic information,therefore,it is very difficult to distinguish them using common genetic markers such as short tandem repeat(STR),single nucleotide polymorphism(SNP)and insertion/deletion(Indel).However,some high variable regions across genome may be possible to record the variants between MZT individuals.For example,DYZ1 array is consist of highly tandem repeats with thousand copies,containing many variants due to complex structure.Some STRs located in Y chromosome show higher mutating rates over than 0.01,named as rapidly mutating(RM)Y-STRs.And mitochondrial genome(mt Genome)also have much higher mutating rate,which is 10-fold of nuclear genome.The mutations across these markers might record rare variants within MZT.Above all,we attempt to study DYZ1 array,RM Y-STR and mt Genome,clarify their possible values in MZT discrimination as well as to build suitable technique methods,providing scientific support for forensic MZT discrimination.Methods:1.We used Q5 ultra-fidelity enzyme to prepare 3.5kb DYZ1 array as templates and then purified them before constructing SMRT libraries,finally detected those DYZ1 libraries of 3 MZT pairs on Pac Bio Sequel platform.Mapping to Y chromosome(NC?000024.10)and to DYZ1 arrays(X06228.1),output reads were analyzed by online multiple alignment tools.2.We used Illumina Ampliseq technology to build a 19 RM Y-STRs NGS panel,including DYF387S1,DYF399S1,DYF403S1 a,DYF403S1b1,DYF403S1b12,DYF404S1,DYS449,DYS518,DYS526 I,DYS526II,DYS547,DYS570,DYS576,DYS612,DYS627,DYS464,DYS527,DYS630 and DYS713.Then,we used it to detect 38 pairs of MZT and 2800 M DNA.Finally,we obtained profiles of all samples via Wintermute software and STRait Razor software,and compared capillary electrophoresis(CE)results and NGS results of each sample as well as RM Y-STR profiles between each MZT pair.3.To build single molecular real-time(SMRT)sequencing mt Genome technology,we adopted REPLI-g Mitochondrial DNA Kit to amplify mt Genome of 16 pairs of MZT from their whole blood sample,and sequenced them on Pac Bio Sequel platform.Using 2% detection threshold,we obtained SMRT generating haplotypes of 16 pairs of MZT in the comparison of PGM haplotypes of same samples.In the end,we screened and analyzed low-level variants of MZT with both 2% and 5% detection threshold.Results:1.DYZ1 arrays generated from SMRT sequencing were accurate and deeply covered,focusing on 7 regions with high coverage.Referring to DYZ1(X06228.1)in Gene Bank,the identity of SMRT results were obviously higher than that of Sanger results.Analyzing DYZ1 array differences within MZT pairs,we found no differences within any MZT pair not matter in 7 regions with deep coverage over than 20 or in DYZ1 consensus.2.RM Y-STR NGS panel was built successfully in the term of good performence in experimental aspect.After analyzing 2800 M DNA,we set 10% as analytical threshold in following analysis.Under 10% threshold,profile of 9947 A,as negative control,showed amplification products only in DYS526 I.Except three loci(DYS713,DYS547,DYS526 II)without complete amplification in core repeat regions,two not included in CE panel(DYS576 and DYS570)and DYS526 I,other 13 loci in NGS profiles of 2800 M DNA were consistent with that in CE profiles.Three detection results of 2800 M by NGS panel were identical completely with well repeatability.Compare to CE results,RM Y-STR NGS results of 38 MZT pairs included not only the same alleles to CE,but also 309 iso-alleles and additional 156 length-based alleles.The total number of additional allele types were 106.In the end,5 distinguishable iso-alleles between 5 MZT pairs were observed.3.The mt Genome results from SMRT sequencing were accurate,repeatable,with deep coverage and good concordance.Under 2% detection threshold and 20 circular consensus sequences(CCSs)reads with Q30 quality,SMRT results showed less corrections and more even coverage pattern than PGM results,as well as none nuclear mitochondrial pseudogenes(NUMTs)accosiated variants,none missing SNP and 917 point heteroplasmies(PHPs)across all samples.Under 2% detection threshold,we found 785 low level variants of 16 pairs of MZT,involving 648 positions.When increase detection threshold to 5%,six MZT pairs still showed rare difference,accounting for 37.5%.Conclusions:In this study,to solve MZT discrimination issue,we first built a SMRT sequencing DYZ1 arrays technology,which is proved convenient,deeply covered,accurate and producible.Using it to detect DYZ1 of 3 MZT pairs,we found no difference between any MZT pair,hinting that DYZ1 might not be an ideal marker for MZT discrimination.We also first developed a 19 RM Y-STRs NGS panel with high specificity,accuracy and repeatability for 15 loci.We used this panel to discover distinguishable iso-alleles between 5 pairs of MZT,indicating this RM Y-STR NGS panel could be a new technical tool to distinguish MZT males.Moreover,we established a long read SMRT mt Genome method,which performed convenient,accurate and reproducible in sequencing.Under 2% detection threshold and at least 20 CCS reads with over Q30 quality,all of the 16 MZT pairs showed many rare differences,indicating that SMRT sequencing mt Genome technology could distinguish MZT.All together,this study provides new markers and develop new techniques to solve forensic MZT discrimination issue.