Tan sheep 50K liquid phase chip based on targeted capture sequencing and application thereof

Abstract

The invention belongs to the technical field of genotyping, and particularly relates to a Tan sheep 50K liquid phase chip based on targeted capture sequencing and application thereof, the Tan sheep 50K liquid phase chip is composed of independently packaged probe mixed liquid and a hybridization capture reagent, the independently packaged probe mixed liquid comprises a Tan sheep 50K probe, and site information of the Tan sheep 50K probe relates to 50000 SNP sites. The prepared Tan sheep 50K liquid chip is mixed with a Tan sheep high-throughput sequencing library, a target site is captured and amplified and purified, and after a product is subjected to high-throughput sequencing, a sequencing result is compared with a sheep reference genome, so that the genetic typing of the Tan sheep to be detected is obtained. The method is applicable to resource evaluation, genetic identification and genome selection of the Tan sheep variety, can break through key technical evaluation restricting genome selection breeding, lays a foundation for building a Tan sheep genome selection sharing platform, and accelerates the realization of the Tan sheep from phenotype selection to genome selection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genotyping, and particularly relates to a 50K liquid phase chip of Tan sheep based on targeted capture sequencing and application thereof. BACKGROUND

[0002] Dual-purpose sheep breeds, such as Tan sheep, play an important role in animal husbandry and economic development. However, in recent years, with the acceleration of scale and intensification of indoor feeding, the inbreeding coefficient of Tan sheep population continues to rise, and the improvement of key economic traits such as growth rate, reproduction rate and feed conversion rate is slow, which has become a bottleneck restricting the quality and efficiency of the industry.

[0003] Molecular marker assisted selection (MAS) and whole genome selection (GS) technologies can complete individual genetic evaluation at an early stage of sheep birth, significantly shorten the generation interval and accelerate genetic progress. The core prerequisite is to obtain high-density genetic markers that are significantly associated with target traits and uniformly cover the whole genome. SNPs are widely distributed in the genome, and are abundant in number and polymorphism. At present, they are widely used in population genetic structure analysis, molecular identity card, genome selection, genetic diversity evaluation, whole genome association analysis and chip preparation. By combining various methods to fully utilize SNP information in the genome, this can lay the foundation for the preparation of SNP chips and provide data sources for mining important sites of economic traits.

[0004] SNP chips can be divided into solid phase chips and liquid phase chips according to the carriers. Compared with solid phase chips, liquid phase chips have the characteristics of high site accuracy, good platform applicability, high detection efficiency, more sites, wide application range and strong flexibility. Liquid phase chips are based on targeted capture sequencing genotyping (GBTS), which can realize deep resequencing of target sites by reducing library abundance, and can significantly reduce the cost of genotyping.

[0005] However, the SNP chips currently developed for Tan sheep still have the problem of limited association with important economic traits of Tan sheep, which is insufficient to cover the genetic markers of important economic traits of Tan sheep. SUMMARY

[0006] To solve the problem in the prior art that the SNP chips currently developed for Tan sheep still have the problem of limited association with important economic traits of Tan sheep, which is insufficient to cover the genetic markers of important economic traits of Tan sheep, the application aims to explore SNP sites related to economic traits of Chinese Tan sheep and prepare chips, so as to play an important role in genetic evaluation of important traits, kinship identification, expansion of purebred population, establishment of pedigree, improvement of breeding value, selection and matching, etc., and provides a 50K liquid phase chip of Tan sheep based on targeted capture sequencing and application thereof. To achieve the above object, the application adopts the following technical scheme.

[0007] Based on the genome resequencing and whole genome association analysis of Tan sheep, the significant associated sites of important economic traits and the detection sites of common Tan sheep economic traits are mined, and a part of background sites are developed, so that a liquid SNP chip for selection, genetic evaluation, molecular marker assisted selection, etc. is imminent. Therefore, the first purpose of the present application is to provide a Tan sheep 50K liquid chip based on targeted capture sequencing, which is composed of independently packaged probe mixtures and hybrid capture reagents.

[0008] The independently packaged probe mixtures comprise probes for detecting 50,000 SNP sites related to Tan sheep economic traits; the 50,000 SNP sites related to Tan sheep economic traits are collectively used for designing the probes, and the target sites are placed in the middle position of the probes.

[0009] The 50,000 SNP sites related to Tan sheep economic traits include 5540 SNP sites on chromosome 1, 5221 SNP sites on chromosome 2, 4701 SNP sites on chromosome 3, 2387 SNP sites on chromosome 4, 2094 SNP sites on chromosome 5, 2631 SNP sites on chromosome 6, 1953 SNP sites on chromosome 7, 1909 SNP sites on chromosome 8, 1831 SNP sites on chromosome 9, 1672 SNP sites on chromosome 10, 1245 SNP sites on chromosome 11, 1566 SNP sites on chromosome 12, 1759 SNP sites on chromosome 13, 1307 SNP sites on chromosome 14, 1655 SNP sites on chromosome 15, 1481 SNP sites on chromosome 16, 1487 SNP sites on chromosome 17, 1316 SNP sites on chromosome 18, 1284 SNP sites on chromosome 19, 1068 SNP sites on chromosome 20, 944 SNP sites on chromosome 21, 977 SNP sites on chromosome 22, 1204 SNP sites on chromosome 23, 863 SNP sites on chromosome 24, 1030 SNP sites on chromosome 25 and 875 SNP sites on chromosome 26 of Tan sheep.

[0010] Among them, the position and variation information of the 50,000 SNP sites related to Tan sheep economic traits are shown in Table 1.

[0011] The application provides a 50K liquid chip based on targeted capture sequencing of Tan sheep, which is composed of independently packaged probe mixtures and hybridization capture reagents, wherein the independently packaged probe mixtures comprise probes for detecting 50,000 SNP sites related to economic traits of Tan sheep; and the 50,000 SNP sites related to economic traits of Tan sheep are collectively used for designing the probes.

[0012] Further, the GC content of the probe is 20% to 80%, the length of the probe is 120 bp, and the 5' end of the probe is modified with a biotin group DNA nucleotide sequence.

[0013] Further, the hybridization capture reagent Library Preparation Kit 1.0、 Library Preparation Kit 1,1.0、 Library Preparation Kit 2、 Universal Adapter System、 Hybridization Probes、 FastHybridization Reagents、 Universal Blockers、 Binding andPurification Beads、 Fast Wash Buffers。

[0014] The second object of the application is to provide the application of the 50K liquid chip of Tan sheep in the analysis of important economic traits of Tan sheep.

[0015] Further, the important economic traits of Tan sheep are at least one selected from body length, body height, chest depth, chest width, chest circumference, pipe circumference, body weight, cross section height, back fat thickness, wool count, shoulder count, flower spike type, tail length, tail width, tail circumference and tail type.

[0016] The third object of the present application is to provide an application of the said Tan sheep 50K liquid chip in Tan sheep genome selection and selective breeding.

[0017] The fourth object of the present application is to provide any one of the applications of the said Tan sheep 50K liquid chip in Tan sheep genetic diversity analysis, Tan sheep kinship identification, Tan sheep breed identification, whole genome association analysis and SNP genotyping detection.

[0018] Further, the method for using the said Tan sheep 50K liquid chip for SNP genotyping detection is as follows:

[0019] Extracting the genomic DNA of the Tan sheep to be tested; using the genomic DNA as a template, constructing a Tan sheep DNA high-throughput sequencing library.

[0020] Using the said Tan sheep 50K liquid chip to hybridize and capture the said Tan sheep DNA high-throughput sequencing library to enrich DNA fragments containing target SNP sites.

[0021] Amplifying and purifying the enriched DNA fragments to obtain an amplification product.

[0022] High-throughput sequencing the said amplification product and aligning the sequencing results with the sheep reference genome ARS-UI_Ramb_v3.0.

[0023] Using the HaplotypeCaller module of the GATK software to generate SNP and InDel variant site files and extract target SNP sites.

[0024] Using 5x filtering and combining the indicators of sequencing depth DP≥5 and quality value GQ≥20 for filtering to obtain SNP genotyping results of high-quality target SNP sites.

[0025] Further, the high-throughput sequencing is performed using the Huada DNBSEQ-T7 sequencing platform.

[0026] Further, the said target SNP sites include functional or representative sites related to important economic traits, genetic diversity, kinship identification or breed identification of Tan sheep.

[0027] Further, the SNP genotyping results of high-quality target SNP sites are used for genetic evaluation, breed identification, kinship identification or genome selection and selective breeding of Tan sheep.

[0028] Compared with the prior art, the present application has the following beneficial effects:

[0029] 1. The present application provides a kind of 50K liquid chip based on target capture sequencing of Tan sheep, which is composed of independently packaged probe mixture and hybrid capture reagent, the independently packaged probe mixture includes probe for detecting 50,000 SNP sites related to economic traits of Tan sheep;The 50,000 SNP sites related to economic traits of Tan sheep are used to design the probe. Among them, the above-mentioned probes are designed for SNP sites significantly related to important economic traits in the whole genome of Tan sheep, which has high specificity and full coverage characteristics;Hybrid capture reagent optimizes reaction conditions, improves hybridization efficiency and stability. The combination of the two realizes the accurate and comprehensive detection of genetic markers of important economic traits of Tan sheep, and further solves the problem that the SNP chip developed for Tan sheep in the prior art still has limited correlation with important economic traits of Tan sheep, so it is not enough to cover the genetic markers of important economic traits of Tan sheep.

[0030] Meanwhile, the 50K liquid chip provided by the present application has the effects of low cost, high detection rate and strong breed specificity, which can solve the problems of few associated sites, low accuracy of genomic selection prediction, high cost, inflexibility and inability to be used in large-scale in existing sheep farms in the prior art.

[0031] 2. The liquid chip according to the present application can mine significant SNP sites for important economic traits of Tan sheep, which are based on Tan sheep population recorded by multidimensional phenotype group, so as to significantly improve the accuracy of genomic breeding value of important economic traits of Tan sheep.

[0032] 3. The liquid chip according to the present application is for whole genome resequencing of a Tan sheep population, which has stronger breed specificity, low detection cost, fast speed and wide application range, and has important significance for selection and breeding of Tan sheep.

[0033] 4. The liquid chip according to the present application is based on target capture sequencing technology, which can obtain more typing information, including target sites and SNP of adjacent target sites. Compared with solid-phase chip, the liquid chip according to the present application has better flexibility, and can adjust target SNP sites by increasing or decreasing probes. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Figure 1 is a diagram showing the distribution of SNP sites on different chromosomes in the 50K liquid chip of Tan sheep according to the present application. DETAILED DESCRIPTION

[0035] The present application will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but should not be understood as limiting the present application. If not specifically stated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specifically stated, can be obtained from commercial channels.

[0036] Example 1: Design and preparation of 50K SNP liquid chip of Tan sheep

[0037] 1. Establishment of Tan sheep SNP database

[0038] The present application utilizes high-depth and low-depth resequencing data of 205 Tan sheep individuals from Hongsefuqu Tiansheng Sheep Breeding and Breeding Co., Ltd., Wuzhong Hongsefuqu Tiansheng Agricultural Science and Technology Development Co., Ltd. and Ningxia Yanchi Tan Sheep Industry Group Suomuxu Breeding Co., Ltd., and ultimately obtains highly reliable SNP sites for subsequent screening by filling low-depth data. The specific process is as follows:

[0039] First, the DNBSEQ-T7 sequencer is used to perform whole genome resequencing on Tan sheep individuals from Hongsefuqu Tiansheng Sheep Breeding and Breeding Co., Ltd., Wuzhong Hongsefuqu Tiansheng Agricultural Science and Technology Development Co., Ltd. and Ningxia Yanchi Tan Sheep Industry Group Suomuxu Breeding Co., Ltd., and the Fastp software is used to quality control the original data with parameters -i-o-I-O, then the BWA software is used to align to the sheep reference genome ARS-UI_Ramb_v3.0, and finally the haplotypecaller module of GATK4 software is used to call SNP for high-depth sequencing individuals, and the combination of BEAGLE v5.4 for phasing and GLIMPSE2 for genotype filling is used to construct a reference panel of 205 Tan sheep high-depth sequencing for filling low-depth data, and a total of 44,763,688 mutation sites are obtained. Sites with a deletion rate > 0.1, a minimum allele frequency < 0.05, and HWE < 0.00001 are filtered out, and SNPs of sex chromosomes are filtered out. After quality control, 18,627,737 SNPs are finally retained for subsequent analysis.

[0040] 2. Screening of functional sites

[0041] For the 16 important economic traits phenotype data collected from the Tan sheep population, first use Excel spreadsheet for pretreatment, eliminate the abnormal value of each trait. After completing the quality control of the phenotype data, using the whole genome association analysis method based on one-step genomic best linear unbiased prediction (ssGBLUP), taking age and gender as fixed effects, using BLUPF90 software to estimate the genomic estimated breeding value (GEBV) of all individuals, and transforming it into SNP effect value. Estimate the variance component by AIREMLF90 module, and then get GEBV and SNP effect value by postGSf90 module, so as to screen out the genomic region significantly related to the target trait, and carry out gene annotation on the genomic region with larger proportion of phenotype explanation. Further compare these significant SNP sites with the known quantitative trait locus (QTL) database (https: / / www.animalgenome.org / cgi-bin / QTLdb / OA / index) to screen out the overlapping region as the candidate functional site.

[0042] 3. Screening of background sites

[0043] According to the principle of uniform distribution of selected SNP sites in genome, the background SNP sites are supplemented on the chip based on resequencing data. The screening principle of background sites is to filter samples and sites with high deletion rate, remove linkage, filter samples with kinship within three generations, filter sites that do not meet Hardy-Weinberg equilibrium rule, filter sites with low allele frequency, and finally perform sliding window selection. The window size is determined according to LD analysis. The sliding window principle is to select an optimal site in each window according to the pre-set priority, such as the site with the largest MAF or the site with the highest priority.

[0044] Through the above steps, a total of 50,000 functional sites and background sites (SNP sites) are selected. According to the 50,000 SNP sites, probe sequences are designed by Beijing Comprehension Agricultural Technology Co., Ltd., high-throughput synthesis of single-stranded DNA probes with biotin modification at the 5' end, Tris-HCl quantification (including 4 μL CAGT probe, 8 μL universal blocking agent and 5 μL blocking agent solution), and finally obtain independently packaged Tan sheep 50K probe mixture. Among them, the independently packaged Tan sheep 50K probe mixture is referred to as independently packaged probe mixture. The 50,000 SNP sites are 50,000 SNP sites related to Tan sheep economic traits.

[0045] The independently packaged probe mixture includes probes for detecting 50,000 SNP sites related to Tan sheep economic traits; the 50,000 SNP sites related to Tan sheep economic traits are used to design probes, and the target site is placed in the middle position of the probe.

[0046] Hybridization capture reagents Library Preparation Kit 1.0, LibraryPreparation Kit 1,1.0, Library Preparation Kit 2, UniversalAdapter System, Hybridization Probes, Fast HybridizationReagents, Universal Blockers, Binding and Purification Beads, Fast Wash Buffers.

[0047] Finally, the Tan sheep 50K liquid chip is composed of independently packaged Tan sheep 50K probe mixture and hybridization capture reagents. When used, they are mixed according to the protocol to complete the liquid hybridization capture, and a set of liquid target capture system is formed.

[0048] Among them, the Tan sheep SNP liquid chip is also called Tan sheep 50K SNP liquid chip.

[0049] The position and variation information of the above-mentioned 50,000 SNP sites are represented in the form of chromosome physical position: reference genotype / variation allele genotype, and the position and variation information of the SNP sites are shown in Tables 1-.

[0050] Table 1 Position and variation information of SNP sites

[0051]

[0052]

[0053] Table 2 Table 1 continuation table 1

[0054]

[0055]

[0056] Table 3 Table 1 continuation table 2

[0057]

[0058]

[0059] Table 4 Table 1 continuation table 3

[0060] Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt 1_40970016: T / C 1_41062904: T / C 1_41096149: C / T 1_41157955: T / C 1_41182277: A / T 1_41239370: A / G 1_41315576: A / G 1_41388420: A / G 1_41410431: T / C 1_41451983: C / A 1_41542890: G / A 1_41579350: T / G 1_41626325: G / A 1_41707199: T / C 1_41750253: G / A 1_41798231: T / C 1_41885205: G / C 1_41930979: A / G 1_41956579: C / T 1_42044085: C / G 1_42108227: G / T 1_42161579: A / G 1_42202043: T / A 1_42258838: A / G 1_42318952: T / G 1_42349889: C / T 1_42426482: G / T 1_42472871: T / C 1_42513405: A / G 1_42588273: T / C 1_42630928: G / A 1_42687769: A / G 1_42713744: G / A 1_42800305: A / T 1_42835546: T / C 1_42898749: C / G 1_42960203: G / T 1_43024206: C / A 1_43064424: T / C 1_43139635: G / A 1_43168427: A / T 1_43254300: G / A 1_43263127: T / C 1_43365807: A / G 1_43421237: A / G 1_43456520: C / G 1_43523109: A / G 1_43571885: T / C 1_43642519: T / C 1_43677154: A / G 1_43739542: G / A 1_43802022: T / G 1_43848476: T / C 1_43873588: T / C 1_43944048: A / G 1_44020856: A / G 1_44056814: A / G 1_44097013: T / C 1_44176157: T / C 1_44205031: C / T 1_44280634: A / C 1_44304676: G / A 1_44371403: T / G 1_44462766: A / G 1_44488952: T / C 1_44548254: G / A 1_44591203: G / A 1_44643942: T / C 1_44718348: T / C 1_44864842: C / T 1_44920267: G / A 1_45002108: T / C 1_45052500: G / A 1_45111216: C / T 1_45168089: T / A 1_45230591: T / C 1_45237179: G / A 1_45336325: C / T 1_45364902: A / G 1_45453934: C / G 1_45478304: A / G 1_45560159: T / A 1_45601278: C / T 1_45624324: A / C 1_45696869: T / G 1_45779851: T / C 1_45798328: G / A 1_45876415: T / C 1_45896461: A / C 1_45967346: G / A 1_46032380: T / C 1_46067314: G / A 1_46116482: A / C 1_46173083: C / T 1_46236425: G / A 1_46320022: G / C 1_46346048: A / T 1_46401489: G / A 1_46455875: T / C 1_46496971: G / C 1_46564239: G / A 1_46624651: A / G 1_46695827: A / G 1_46721827: C / G 1_46783736: A / G 1_46829293: C / G 1_46914786: C / T 1_46986324: A / T 1_47038087: G / A 1_47078582: G / A 1_47133776: T / C 1_47160354: T / G 1_47218054: C / T 1_47265580: C / T 1_47318751: G / A 1_47395728: C / T 1_47466999: C / T 1_47487437: C / A 1_47577969: A / C 1_47644995: C / T 1_47664404: A / G 1_47723923: C / T 1_47785847: C / T 1_47853312: A / G 1_47902950: T / C 1_47962626: A / C 1_48026119: T / C 1_48039941: A / G 1_48127059: A / G 1_48194570: G / A 1_48214483: T / C 1_48297551: A / C 1_48324692: C / G 1_48384146: A / T 1_48464767: C / T 1_48471300: G / T 1_48569273: T / G 1_48597508: A / C 1_48635456: A / G 1_48782622: T / C 1_48799160: G / A 1_48878206: T / G 1_48930009: A / C 1_449008052: G / A 1_49071760: A / G 1_49107449: G / A 1_49177303: A / G 1_49206495: T / G 1_49279909: A / G 1_49346155: G / A 1_49358107: C / T 1_49407511: T / C 1_49508930: T / A 1_49530373: G / A 1_49614376: G / C 1_49630455: C / T 1_49720548: A / T 1_49770125: T / C 1_49813976: C / T 1_49841173: G / A 1_49941326: G / A 1_49959471: A / T 1_50031619: T / C 1_50090346: G / T 1_50139607: A / G 1_50218730: G / C 1_50229265: T / C 1_50239644: T / C 1_50239656: T / C 1_50252928: T / C 1_50256198: G / T 1_50261924: G / A 1_50262725: G / A 1_50272267: T / G 1_50273263: C / G 1_50282110: C / A 1_50288477: G / A 1_50301794: T / C 1_50301925: C / A 1_50302398: T / C 1_50339741: A / G 1_50417903: C / T 1_50481353: C / T 1_50531123: A / G 1_50603291: A / G 1_50659977: G / T 1_50689440: T / C 1_50768296: A / G 1_50803843: A / G 1_50845262: A / T 1_50901612: G / A 1_50955389: A / G 1_50998737: T / G 1_51057685: C / T 1_51130299: C / T 1_51170394: A / C 1_51237483: C / T 1_51281132: G / A 1_51322293: T / C 1_51392923: A / G 1_51442776: C / T 1_51497501: T / C 1_51544906: G / A 1_51617169: A / G 1_51701611: C / T 1_51735296: G / A 1_51809155: T / C 1_51843689: T / C 1_51918717: C / T 1_51937323: C / T 1_52027960: C / T 1_52067869: A / C 1_52121549: T / C 1_52156938: A / G 1_52225387: A / G 1_52238215: T / C 1_52242303: G / A 1_52256794: A / C 1_52269123: G / A 1_52270877: A / G 1_52271010: A / G 1_52350246: G / T 1_52395683: A / G 1_52420503: G / T 1_52525924: A / G 1_52560133: A / T 1_52633252: G / A 1_52672660: A / T 1_52708549: C / T 1_52795807: C / A 1_52841921: G / T 1_52882688: A / G 1_52946556: A / G 1_52990898: T / C 1_53035126: T / A 1_53121981: G / A 1_53135550: C / T 1_53229507: A / G 1_53282437: A / G 1_53310536: A / T 1_53360560: C / A 1_53431732: C / T 1_53500631: A / G 1_53527631: C / T 1_53608398: G / A 1_53647300: A / C 1_53732027: A / C 1_53783485: T / C 1_53806572: G / A 1_53862261: C / T 1_53904877: G / A 1_53991320: T / C 1_54011567: T / C 1_54109388: T / G 1_54136210: C / A 1_54208099: A / G 1_54265781: A / G 1_54329015: T / A 1_54371406: C / G 1_54446149: C / A 1_54465198: G / A 1_54544054: A / G 1_54586879: A / G 1_54660112: G / C 1_54714640: A / T 1_54786949: G / A 1_54880967: C / T 1_54938541: A / T 1_54984481: G / T 1_55033992: T / C 1_55062068: C / T 1_55158593: A / G 1_55171221: A / C 1_55238368: T / C 1_55308537: C / T Chr_Pos: Ref / Alt

[0061] Table 5 continuation of Table 1 4

[0062] ​ Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt 1_55338722: C / T 1_55412403: A / G 1_55452338: T / C 1_55531273: C / T 1_55589908: G / A 1_55610143: T / C 1_55689256: C / G 1_55759153: T / C 1_55815071: A / G 1_55835898: G / A 1_55907857: G / C 1_55972281: T / C 1_56031949: G / T 1_56074042: A / G 1_56135064: C / A 1_56152052: T / C 1_56203380: C / A 1_56256890: C / T 1_56343686: C / T 1_56371281: G / A 1_56455694: T / G 1_56515734: A / G 1_56571596: T / A 1_56592613: A / T 1_56668718: A / G 1_56736480: G / A 1_56784809: C / T 1_56821959: C / T 1_56910990: A / G 1_56956372: G / A 1_56979235: C / T 1_56979522: A / G 1_56979837: G / A 1_56981862: G / A 1_56983358: T / C 1_56985069: A / C 1_56985240: A / C 1_56985505: C / G 1_56985667: T / A 1_56985933: T / A 1_56986310: C / A 1_56987721: G / C 1_57023424: G / T 1_57027352: C / A 1_57036387: A / G 1_57037991: T / C 1_57038399: T / C 1_57041767: C / T 1_57043056: C / T 1_57043992: C / / T 1_57044603: C / T 1_57045977: G / A 1_57048188: C / T 1_57051332: T / C 1_57053619: T / A 1_57057217: T / C 1_57057359: G / A 1_57058398: C / T 1_57058411: G / C 1_57058526: T / C 1_57099634: T / C 1_57133238: A / G 1_57226335: A / G 1_57279027: G / C 1_57312035: G / A 1_57354912: T / C 1_57438801 : C / T 1_57542806 : G / A 1_57588578 : C / T 1_57657582 : C / G 1_57719600 : T / C 1_57759541 : C / T 1_57839615 : T / G 1_57894536 : G / A 1_57925147 : G / A 1_57963912 : C / G 1_58062875 : A / G 1_58116856 : C / A 1_58171120 : T / C 1_58205545 : A / C 1_58258777 : A / C 1_58334447 : C / T 1_58383399 : G / A 1_58405871 : A / G 1_58483701 : C / G 1_58520185 : A / G 1_58597322 : A / G 1_58620686 : C / T 1_58668346 : A / C 1_58776460 : A / C 1_58811922 : A / G 1_58859977 : T / C 1_58895764 : G / C 1_58970640 : A / C 1_59042712 : A / G 1_59054760 : T / C 1_59145616 : G / A 1_59187349 : G / T 1_59248435 : A / G 1_59282346 : C / T 1_59370422 : C / T 1_59410868 : A / G 1_59457294 : G / A 1_59511717 : T / C 1_59588829 : C / T 1_59639272 : C / T 1_59699752 : G / A 1_59714800 : A / G 1_59781736 : A / G 1_59855429 : C / T 1_59896661 : C / T 1_59976831 : A / T 1_60004703 : A / G 1_60086234 : T / A 1_60103141 : T / G 1_60199681 : G / A 1_60238126 : G / A 1_60280411 : C / T 1_60341665 : T / C 1_60405045 : A / T 1_60442111 : A / G 1_60511316 : G / A 1_60576448 : C / T 1_60617669 : A / T 1_60654814 : G / C 1_60749290 : T / C 1_60796796 : T / C 1_60841157 : T / C 1_60885211 : T / C 1_60959429 : T / C 1_60992482 : A / G 1_61042254 : G / A 1_61114429 : T / C 1_61151276 : T / C 1_61208585 : T / G 1_61271980 : A / G 1_61309189 : G / T 1_61342589 : G / A 1_61376044 : G / A 1_61409589 : G / A 1_61443034 : G / A 1_61476579 : G / A 1_61510124 : G / A 1_61543669 : G / A 1_61577114 : G / A 1_61610559 : G / A 1_61644004 : G / A 1_61677449 : G / A 1_61710894 : G / A 1_61744339 : G / A 1_61777784 : G / A 1_61811229 : G / A 1_61844674 : G / A 1_61878119 : G / A 1_61911564 : G / A 1_61945009 : G / A 1_61978454 : G / A 1_62011899 : G / A 1_62045344 : G / A 1_62078789 : G / A 1_62112234 : G / A 1_62145679 : G / A 1_62179124 : G / A 1_62212569 : G / A 1_62246014 : G / A 1_62279459 : G / A 1_62312894 : G / A 1_62346339 : G / A 1_62379784 : G / A 1_62413229 : G / A 1_62446674 : G / A 1_62480119 : G / A 1_62513564 : G / A 1_62547009 : G / A 1_62580454 : G / A 1_62613899 : G / A 1_62647344 : G / A 1_62680789 : G / A 1_62714234 : G / A 1_62747679 : G / A 1_62781124 : G / A 1_62814569 : G / A 1_62848014 : G / A 1_62881559 : G / A 1_62915004 : G / A 1_62948449 : G / A 1_62981894 : G / A 1_63015339 : G / A 1_63048784 : G / A 1_63082229 : G / A 1_63115674 : G / A 1_63149119 : G / A 1_63182564 : G / A 1_63216009 : G / A 1_63249454 : G / A 1_63282899 : G / A 1_63316344 : G / A 1_63349789 : G / A 1_63383234 : G / A 1_63416679 : G / A 1_63450124 : G / A 1_63483569 : G / A 1_63517014 : G / A 1_63550459 : G / A 1_63583894 : G / A 1_63617339 : G / A 1_63650784 : G / A 1_63684229 : G / A 1_63717674 : G / A 1_63751119 : G / A 1_63784564 : G / A 1_63818009 : G / A 1_63851454 : G / A 1_63884899 : G / A 1_63918344 : G / A 1_63951789 : G / A 1_63985234 : G / A 1_64018679 : G / A 1_64052124 : G / A 1_64085569 : G / A 1_64119014 : G / A 1_64152459 : G / A 1_64185894 : G / A 1_64219339 : G / A 1_64252784 : G / A 1_64286229 : G / A 1_64319674 : G / A 1_64353119 : G / A 1_64386564 : G / A 1_64420009 : G / A 1_64453454 : G / A 1_64486899 : G / A 1_64520344 : G / A 1_64553789 : G / A 1_64587234 : G / A 1_64620679 : G / A 1_64654124 : G / A 1_64687569 : G / A 1_64721014 : G / A 1_64754459 : G / A 1_64787894 : G / A 1_64821339 : G / A 1_64854784 : G / A 1_64888229 : G / A 1_64921674 : G / A 1_64955119 : G / A 1_64988564 : G / A 1_65022009 : G / A 1_65055454 : G / A 1_65088899 : G / A 1_65122344 : G / A 1_65155789 : G / A 1_65189234 : G / A 1_65222679 : G / A 1_65256124 : G / A 1_65289569 : G / A 1_65323014 : G / A 1_65356459 : G / A 1_65389894 : G / A 1_65423339 : G / A 1_65456784 : G / A 1_65490229 : G / A 1_65523674 : G / A 1_65557119 : G / A 1_65590564 : G / A 1_65624009 : G / A 1_65657454 : G / A 1_65690899 : G / A 1_65724344 : G / A 1_65757789 : G / A 1_65791234 : G / A 1_65824679 : G / A 1_65858124 : G / A 1_65891569 : G / A 1_65925014 : G / A 1_65958459 : G / A 1_65991894 : G / A 1_66025339 : G / A 1_66058784 : G / A 1_66092229 : G / A 1_66125674 : G / A 1_66159119 : G / A 1_66192564 : G / A 1_66226009 : G / A 1_66259454 : G / A 1_66292899 : G / A 1_66326344 : G / A 1_66359789 : G / A 1_66393234 : G / A 1_66426679 : G / A 1_66460124 : G / A 1_66493569 : G / A 1_66923014 : G / A 1_66956459 1_61398700: A / G 1_61423379: C / T 1_61509494: T / C 1_61536525: G / T 1_61614818: G / A 1_61678056: T / C 1_61718615: A / G 1_61771048: A / T 1_61810084: C / T 1_61898979: C / T 1_61955192: G / A 1_61991650: T / G 1_62017151: C / T 1_62073878: A / G 1_62137895: A / G 1_62226937: A / G 1_62233829: T / G 1_62317779: G / A 1_62363856: T / G 1_62449184: T / C 1_62459758: A / T 1_62507210: C / T 1_62570546: T / G 1_62631090: G / A 1_62724992: A / G 1_62739942: C / T 1_62794568: G / A 1_62886896: G / A 1_62906375: G / C 1_62963149: C / G 1_63034941: C / A 1_63100940: A / G 1_63164116: A / G 1_63207240: C / T 1_63252966: T / C 1_63309541: A / G 1_63342402: C / T 1_63430934: C / T 1_63471483: C / T 1_63526735: C / T 1_63593400: A / G 1_63625476: G / C 1_63664843: A / G 1_63747339: T / C 1_63810150: T / C 1_63851186: G / A 163858447: G / A 1_63858988: C / A 1_63859508: C / T 1_63861843: G / A 1_63868081: C / T 1_63870317: A / G 1_63875607: G / C 1_63882836: A / G 1_63882895: T / C 1_63885037: C / T 1_63888325: A / G 1_63890465: G / T 1_63890964: G / T 1_63894873: G / A 1_63899162: G / A 1_63900322: C / G 1_63905655: T / C 1_63906722: G / T 1_63962219: A / G 1_63990932: T / A 1_64087961: A / G 1_64150028: G / T 1_64157958: C / T 1_64234683: A / G 1_64264020: G / A 1_64317442: T / C 1_64418192: G / A 1_64432562: C / T 1_64514174: A / G 1_64550139: T / C 1_64660761: T / C 1_64652112: T / C 1_64715137: T / C 1_64794517: T / G 1_64812349: G / A 1_64879467: T / G 1_64920846: G / A 1_65021418: C / G 1_65055272: A / G 1_65129606: A / G 1_65155118: T / C 1_65204072: T / C 1_65293638: T / G 1_65328634: T / A 1_65406182: G / T 1_65436574: C / A 1_65509382: G / A 1_65525499: C / G 1_65628294: G / A 1_65657572: T / G 1_65709886: C / G 1_65784205: T / C 1_65824935: A / G 1_65875888: A / T 1_65933810: C / G 1_65988939: G / T 1_66027432: G / T 1_66072887: G / T 1_66158190: A / G 1_66189715: G / A 1_66259228: T / C 1_66311690: G / A 1_66360925: T / C 1_66406106: C / T 1_66501964: T / A 1_66552915: T / A 1_66580691: T / A 1_66661919: G / A 1_66674331: T / C 1_66781613: C / T 1_66797439: A / G 1_66797611: C / G 1_66797627: T / G 1_66799786: A / T 1_66802343: A / G 1_66803010: A / G 1_66803452: G / T 1_66807511: G / A 1_66807628: T / G 1_66807990: G / C 1_66811441: G / A 1_66811777: G / C 1_66812094: T / C 1_66814975: A / G 1_66817016: T / A 1_66818019: G / A 1_66818188: A / C 1_66824541: T / C 1_66828337: C / T 1_66835734: C / T 1_66839477: A / C 1_66840877: C / T 1_66852070: T / A 1_66852783: C / G 1_66853776: A / G 1_66899428: G / A 1_66963196: C / T 1_67043631: C / T 1_67076560: T / G

[0063] Table 6 continuation of Table 5

[0064] Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt 1_67160732: T / C 1_67209887: G / C 1_67240014: C / T 1_67314647: C / T 1_67365139: T / C 1_67436438: C / G 1_67479230: T / G 1_67542721: G / A 1_67658112: C / T 1_67692627: C / T 1_67764522: T / A 1_67782854: G / A 1_67786799: A / G 1_67801570: A / G 1_67801570: A / G 1_67801570: A / G 1_67808458: C / T 1_67809632: T / G 1_67810141: T / G 1_67854105: A / G 1_67892004: G / A 1_68015423: G / T 1_68055922: G / T 1_68141156: A / T 1_68191137: T / G 1_68221207: C / T 1_68292423: C / T 1_68292639: T / C 1_68298360: T / C 1_68299659: C / T 1_68303521: C / T 1_68305521: A / G 1_68307324: C / T 1_68309110: T / A 1_68312561: T / C 1_68314896: G / A 1_68317891: C / T 1_68319103: C / T 1_68319701: T / C 1_68328778: C / T 1_68332800: T / G 1_68351553: A / G 1_68354009: C / A 1_68356748: C / G 1_68359107: G / T 1_68359174: T / C 1_68359300: A / G 1_68419657: T / A 1_68476558: C / T 1_68528084: T / C 1_68567766: A / T 1_68651689: T / C 1_68724340: A / G 1_68796714: A / G 1_68855732: G / A 1_68913275: C / A 1_68959249: G / A 1_68983859: C / T 1_69079984: A / G 1_69117183: C / A 1_69167940: T / C 1_69236670: A / G 1_69297754: C / T 1_69317653: T / G 1_69387093: C / T 1_69442685: C / T 1_69484499: G / A 1_69538776: G / A 1_69619581: A / G 1_69657367: G / A 1_69726688: G / A 1_69768631: T / C 1_69816681: C / T 1_69874330: A / T 1_69926987: T / C 1_69968243: G / A 1_70020768: G / A 1_70104854: A / G 1_70132917: A / G 1_70188062: A / T 1_70244522: A / T 1_70310167: A / G 1_70380285: T / G 1_70419618: T / C 1_70457210: G / A 1_70528721: A / T 1_70573535: G / T 1_70627231: A / C 1_70686036: G / A 1_70779216: C / G 1_70809355: A / G 1_70879573: G / A 1_70903799: C / T 1_70963560: C / T 1_71045844: T / C 1_71107515: T / C 1_71122162: A / T 1_71203039: C / T 1_71242622: T / C 1_71302965: A / G 1_71387215: G / A 1_71406920: A / G 1_71474740: A / G 1_71550662: G / T 1_71593867: C / G 1_71609111: T / C 1_71680630: T / C 1_71736903: T / C 1_71787985: G / A 1_71851282: G / A 1_71900643: C / A 1_71986838: G / A 1_72006837: C / T 1_72066087: T / A 1_72131203: A / G 1_72190160: A / G 1_72212482: A / G 1_72293743: C / T 1_72349983: T / C 1_72411479: A / G 1_72439960: C / T 1_72498773: G / A 1_72562449: T / G 1_72611876: A / G 1_72682938: C / T 1_72755136: A / G 1_72761305: A / G 1_72831874: A / G 1_72886180: A / G 1_72937231: G / A 1_72939636: G / A 1_72943171: T / C 1_72946720: G / A 1_72951747: C / A 1_72955712: T / G 1_72955748: A / G 1_72962488: A / G 1_72969017: G / T 1_72991008: T / C 1_73093357: A / G 1_73185316: A / C 1_73209543: A / G 1_73300924: A / T 1_73324896: T / C 1_73365513: A / G 1_73444811: G / A 1_73519665: A / C 1_73568215: C / A 1_73583559: T / C 1_73676324: C / / T 1_73735791: C / T 1_73754141: C / T 1_73849369: G / C 1_73873591: A / G 1_73957744: T / A 1_74004039: A / G 1_74048834: A / C 1_74126287: A / T 1_74183658: G / A 1_74212108: T / C 1_74293706: A / G 1_74333696: C / T 1_74365297: C / G 1_74436118: G / A 1_74489107: C / T 1_74554663: A / G 1_74596633: T / G 1_74672078: G / T 1_74686091: A / G 1_74745699: G / A 1_74798344: A / G 1_74868519: C / T 1_74898073: A / G 1_74995862: C / / T 1_75055537: T / G 1_75090179: G / A 1_75136638: G / A 1_75176811: T / C 1_75276923: T / C 1_75296273: T / C 1_75387450: C / A 1_75422367: G / C 1_75500000: G / A 1_75513630: C / A 1_75659899: T / G 1_75690041: C / T 1_75729871: T / C 1_75811454: G / T 1_75852619: A / C 1_75898864: A / C 1_75947973: T / A 1_76001247: G / T 1_76073992: A / T 1_76157354: A / G 1_76194932: C / T 1_76229643: G / T 1_76344904: A / T 1_76417471: G / A 1_76476579: A / G 1_76491235: T / C 1_76555249: T / A 1_76641407: A / G 1_76697216: A / G 1_76741944: C / A 1_76772971: T / C 1_76867198: T / A 1_76884315: C / T 1_76948817: T / C 1_77008682: A / G 1_77090516: A / G 1_77112429: C / A 1_77196735: G / A 1_77210966: C / A 1_77306826: T / C 1_77339967: A / G 1_77395622: G / T 1_77424906: C / T 1_77478849: G / A 1_77537649: G / C 1_77610530: G / A 1_77684828: C / T 1_77714011: A / G 1_77754639: A / C 1_77840173: G / A 1_77868149: T / C 1_77958246: T / C 1_77970640: G / A 1_78061573: A / G 1_78111889: C / A 1_78184530: A / G 1_78205665: C / T 1_78249513: C / G 1_78330743: G / A 1_78388876: A / C 1_78421689: T / C 1_78500625: C / T 1_78518157: A / G 1_78618050: T / A 1_68659336: C / T 1_78697178: G / A 1_78771484: T / C 1_78818866: A / G 1_78874033: A / T 1_78909559: C / T 1_78989427: C / G 1_79061843: A / G 1_79102754: A / G 1_79130438: A / T 1_79201619: G / A 1_79239541: T / G 1_79332602: T / G 1_79361151: C / T 1_79413198: C / T / t 1_79468080: A / G 1_79517476: T / C 1_79601167: G / C 1_79652953: C / T 1_79718371: T / G 1_79768620: T / C 1_79814479: C / T 1_79868087: A / G 1_79917365: C / T 1_79992432: C / T 1_80028000: G / A 1_80064289: T / C 1_80158184: A / G 1_80188126: A / G 1_80219501: C / T 1_80318534: A / G 1_80342269: A / G 1_80425031: A / T 1_80486878: A / C 1_80514267: G / A 1_80547181: T / C 1_80620069: C / G 1_80690904: C / A 1 80754973: A / G

[0065] Table 7 continuation of Table 6

[0066]

[0067]

[0068] Table 8 continuation of Table 7

[0069]

[0070]

[0071] Table 9 continuation of Table 8

[0072]

[0073]

[0074] Table 10 continuation of Table 9

[0075]

[0076]

[0077] Table 11 continuation of Table 10

[0078]

[0079]

[0080] Table 12 continuation of Table 11

[0081]

[0082]

[0083] Table 13 continuation of Table 12

[0084]

[0085]

[0086] Table 14 continuation of Table 13

[0087]

[0088]

[0089] Table 15 Table 1 continued 14

[0090]

[0091]

[0092] Table 16 Table 1 continued 15

[0093]

[0094]

[0095] Table 17 Table 1 continued 16

[0096]

[0097]

[0098] Table 18 Table 1 continued 17

[0099]

[0100]

[0101] Table 19 Table 1 continued 18

[0102]

[0103]

[0104] Table 20 Table 1 continued 19

[0105]

[0106]

[0107] Table 21 Table 1 continued 20

[0108]

[0109]

[0110] Table 22 Table 1 continued 21

[0111]

[0112]

[0113] Table 23 continuation of Table 1 22

[0114]

[0115]

[0116] Table 24 continuation of Table 1 23

[0117]

[0118]

[0119] Table 25 continuation of Table 1 24

[0120]

[0121]

[0122] Table 26 continuation of Table 1 25

[0123]

[0124]

[0125] Table 27 continuation of Table 1 26

[0126]

[0127]

[0128] Table 28 continuation of Table 1 27

[0129]

[0130]

[0131] Table 29 continuation of Table 1 28

[0132]

[0133]

[0134] Table 30 continuation of Table 1 29

[0135]

[0136]

[0137] Table 31 continuation of Table 1 30

[0138]

[0139]

[0140] Table 32 continuation of Table 1

[0141]

[0142]

[0143] Table 33 continuation of Table 1

[0144]

[0145]

[0146] Table 34 continuation of Table 1

[0147]

[0148]

[0149] Table 35 continuation of Table 1

[0150]

[0151]

[0152] Table 36 continuation of Table 1

[0153]

[0154]

[0155] Table 37 continuation of Table 1

[0156]

[0157]

[0158] Table 38 continuation of Table 1

[0159]

[0160]

[0161] Table 39 continuation of Table 1

[0162]

[0163]

[0164] Table 40 continuation of Table 1

[0165]

[0166]

[0167] Table 41 continuation of Table 1 40

[0168]

[0169]

[0170] Table 42 continuation of Table 1 41

[0171]

[0172]

[0173] Table 43 continuation of Table 1 42

[0174]

[0175]

[0176] Table 44 continuation of Table 1 43

[0177]

[0178]

[0179] Table 45 continuation of Table 1 44

[0180]

[0181]

[0182] Table 46 continuation of Table 1 45

[0183]

[0184]

[0185] Table 47 continuation of Table 1 46

[0186]

[0187]

[0188] Table 48 continuation of Table 1 47

[0189]

[0190]

[0191] Table 49 continuation of Table 1 48

[0192]

[0193]

[0194] Table 50 Table 1 continued from Table 49

[0195]

[0196]

[0197] Table 51 Table 1 continued from Table 50

[0198]

[0199]

[0200] Table 52 Table 1 continued from Table 51

[0201]

[0202]

[0203] Table 53 Table 1 continued from Table 52

[0204]

[0205]

[0206] Table 54 Table 1 continued from Table 53

[0207]

[0208]

[0209] Table 55 Table 1 continued from Table 54

[0210]

[0211]

[0212] Table 56 Table 1 continued from Table 55

[0213]

[0214]

[0215] Table 57 Table 1 continued from Table 56

[0216]

[0217]

[0218] Table 58 continuation of Table 1 57

[0219]

[0220]

[0221] Table 59 continuation of Table 1 58

[0222]

[0223]

[0224] Table 60 continuation of Table 1 59

[0225]

[0226]

[0227] Table 61 continuation of Table 1 60

[0228]

[0229]

[0230] Table 62 continuation of Table 1 61

[0231]

[0232]

[0233] Table 63 continuation of Table 1 62

[0234]

[0235]

[0236] Table 64 continuation of Table 1 63

[0237]

[0238]

[0239] Table 65 continuation of Table 1 64

[0240]

[0241]

[0242] Table 66 continuation of Table 1 65

[0243]

[0244]

[0245] Table 67 continuation of Table 1 66

[0246]

[0247]

[0248] Table 68 continuation of Table 1 67

[0249]

[0250]

[0251] Table 69 continuation of Table 1 68

[0252]

[0253]

[0254] Table 70 continuation of Table 1 69

[0255]

[0256]

[0257] Table 71 continuation of Table 1 70

[0258]

[0259]

[0260] Table 72 continuation of Table 1 71

[0261]

[0262]

[0263] Table 73 continuation of Table 1 72

[0264]

[0265]

[0266] Table 74 continuation of Table 1 73

[0267]

[0268]

[0269] Table 75 continuation of Table 1 74

[0270]

[0271]

[0272] Table 75 continuation of Table 1

[0273]

[0274]

[0275] Table 76 continuation of Table 1

[0276]

[0277]

[0278] Table 77 continuation of Table 1

[0279]

[0280]

[0281] Table 78 continuation of Table 1

[0282]

[0283]

[0284] Table 79 continuation of Table 1

[0285]

[0286]

[0287] Table 80 continuation of Table 1

[0288]

[0289]

[0290] Table 81 continuation of Table 1

[0291]

[0292]

[0293] Table 82 continuation of Table 1

[0294]

[0295]

[0296] Table 83 continuation of Table 1

[0297]

[0298]

[0299] Table 85 Table 1 continued from Table 84

[0300]

[0301]

[0302] Table 86 Table 1 continued from Table 85

[0303]

[0304]

[0305] Table 87 Table 1 continued from Table 86

[0306]

[0307]

[0308] Table 88 Table 1 continued from Table 87

[0309]

[0310]

[0311] Table 89 Table 1 continued from Table 88

[0312]

[0313]

[0314] Table 90 Table 1 continued from Table 89

[0315]

[0316]

[0317] Table 91 Table 1 continued from Table 90

[0318]

[0319]

[0320] Table 92 Table 1 continued from Table 91

[0321]

[0322]

[0323] Table 93 Table 1 continued from Table 92

[0324]

[0325]

[0326] Table 94 continuation of Table 1

[0327]

[0328]

[0329] Table 95 continuation of Table 1

[0330]

[0331]

[0332] Table 96 continuation of Table 1

[0333]

[0334]

[0335] Table 97 continuation of Table 1

[0336]

[0337]

[0338] Table 98 continuation of Table 1

[0339]

[0340]

[0341] Table 99 continuation of Table 1

[0342]

[0343]

[0344] Table 100 continuation of Table 1

[0345] Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt 9 62227188: T / C 9 62267820: T / C 9 62331264: A / C 9 62368402: A / T 9 62405961: A / C 9 62456369: G / A 9 62538002: T / C 9 62578338: G / A 9 62667710: A / C 9 62680576: C / T 9 62778979: T / C 9 62792863: A / G 9 62837486: T / C 9 62935956: A / C 9-62972051: C / T 9-63049940: C / A 9 63072111: C / A 9 63139486: A / G 9 63209360: T / C 9 63259782: A / C 9 63273910: C / T 9 63346181: G / A 9 63400896: A / C 9 63448128: C / T 9-63536451: A / C 9 63569660: T / C 9-63655523: A / C 9 63706479: T / A 9 63733314: T / G 9 63778851: C / T 9 63851787: G / A 9 63925507: C / T 9 63945616: T / G 9 64033032: G / C 9 64086268: C / T 9 64203894: C / T 9 64239474: T / G 9 64307802: T / C 9 64330708: G / A 9 64381861: A / C 9 64437420: C / T 9 64532214: C / A 9 64539565: C / T 9 64598058: T / G 9 64647075: G / A 9 64701263: G / A 9 64783027: A / G 9 64834179: A / T 9 64874920: G / A 9 64946765: A / G 9 65027550: G / A 9 65063688: C / T 9 65088442: A / C 9 65182242: A / G 9 65220977: A / G 9 65298566: C / A 9 65314611: G / T 9 65385316: G / A 9 65442985: A / G 9 65520014: A / G 9 65563944: G / A 9-65602817: T / C 9 65679432: A / G 9 65699838: C / T 9_65792361: C / A 9_65845439: C / T 9_65865634: G / C 9_65934432: A / G 9_65987346: G / A 9_66020173: G / T 9_66095353: C / T 9_66177534: T / A 9_66194936: T / A 9_66261835: C / T 9_66322661: G / A 9_66360660: A / T 9_66453667: G / T 9-66495556: G / A 9_66523707: T / G 9_66613178: C / T 9_66657818: T / C 9_66716947: A / C 9_66741919: G / C 9_66835065: G / A 9_66879387: C / T 9_66901224: T / C 9_66966156: T / C 9_67016526: G / A 9_67076590: G / A 9_67162256: C / T 9_67203477: A / G 9_67221795: G / A 9_67325021: A / G 9_67372418: A / G 9_67436577: T / C 9_67453559: G / C 9_67504887: A / G 9_67579357: G / A 9_67647351: A / G 9_67704265: C / G 9_67743902: C / T 9_67806735: C / T 9_67868211: A / G 9_67925653: A / T 9_67954976: A / G 9_68028614: G / A 9_68069826: T / C 9_68132312: A / G 9_68185713: A / T 9_68217298: A / G 9_68265288: A / G 9_68338780: C / T 9_68406078: G / A 9_68432878: G / C 9_68490040: A / G 9_68573276: A / T 9_68603619: T / C 9_68673544: A / G 9_68735213: A / G 9_68790059: A / G 9_68814807: G / A 9_68918828: T / C 9_68969778: G / C 9_69003076: T / G 9_69044130: A / T 9_69132790: T / A 9_69176875: C / T 9_69233544: A / G 9_69261356: G / A 9_69359299: T / C 9_69384323: C / T 9_69440322: T / G 9_69518784: C / T 9_69535663: G / A 9_69628782: T / G 9_69664240: T / C 9_69705789: T / G 9_69747281: G / A 9_69845140: C / T 9_69871350: G / A 9_69944667: G / A 9_70004878: A / C 9_70022596: A / T 9_70103161: A / G 9_70146239: T / C 9_70222425: T / C 9_70238809: G / C 9_70338627: A / G 9_70383024: T / C 9_70410871: A / G 9_70462540: G / C 9_70538547: G / A 9_70605691: T / C 9_70637624: A / G 9_70718265: T / C 9_70769635: G / A 9_70822838: T / C 9_70877906: A / G 9_70924676: G / T 9_71000343: T / C 9_71043972: A / G 9_71109515: G / A 9_71145276: G / C 9_71203431: C / G 9_71261927: G / A 9_71302237: T / G 9_71378968: C / T 9_71437241: A / C 9_71466126: A / T 9_71536261: C / T 9_71604062: G / A 9_71706592: G / A 9_71753690: C / T 9_71792832: G / A 9_71878351: T / C 9_71894753: G / A 9_71965968: T / C 9_72036782: A / G 9_72078256: A / G 9_72151295: T / C 9_72208351: G / A 9_72233568: G / A 9_72311410: G / A 9_72372707: T / C 9_72412088: T / C 9_72481807: G / C 9_72533633: A / G 9_72579520: A / G 9_72619109: C / A 9_72669433: T / G 9_72733272: C / T 9_72772916: C / G 9_72849217: A / G 9_72891066: T / A 9_72934865: A / C 9_73018021: G / A 9_73071942: G / T 9_73105664: T / G 9_73169615: C / A 9_73206840: A / T 9_73265380: C / T 9_73357551: C / T 9_73383454: G / A 9_73469886: G / A 9_73526804: A / C 9_73545508: T / G 9_73619853: A / G 9_73666371: G / A 9_73735180: G / A 9_73753696: T / A 9_73855684: A / G 9_73856820: C / T 9_73916539: T / A 9_73972336: G / A 9_74066623: C / T 9_74114494: T / C 9_74136061: C / T 9_74138780: T / C 9_74139870: G / T 9_74140076: A / G 9_74149044: A / G 9_74155230: T / G 9_74155738: T / C 9_74156615: A / G 9_74159362: T / C 9_74160230: G / A 9_74160555: A / G 9_74160621: C / T 9_74167786: A / C 9_74171211: G / A 9_74176808: G / A 9_74225953: C / T 9_74254989: T / G 9_74298276: A / G 9_74401446: G / A 9_74450292: A / G 9_74465405: A / C 9_74529226: G / A 9_74609171: T / C 9_74669483: A / G 9_74688743: T / C 9_74761326: G / A 9_74826152: C / T 9_74890099: A / G 9_74910094: G / A 9_74978626: G / A 9_75025211: T / C 9_75104573: A / G 9_75124092: G / A 9_75194627: A / G 9_75249841: G / A 9_75313121: C / T 9_75347539: A / G 9_75419753: G / A 9_75485014: C / T 9_75535627: G / A 9_75535641: C / A 9_75537840: A / G 9_75542559: A / G 9_75544111: G / A 9_75547936: T / G 9_75548369: C / T 9_75551567: T / C 9_75551681: T / C 9_75552722: A / C 9_75559148: T / C 9_75662410: T / G 9_75710634: C / T 9_75744381: A / G 9_75793981: T / C 9_75839199: A / C 9_75932175: A / T 9_75980276: G / A 9_76033591: C / T 9_76071112: T / C 9_76121048: A / G

[0346] Table 101 continuation of Table 1

[0347] Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt 9_76201822: C / T 9_76265637: T / C 9_76315984: G / A 9_76339136: C / T 9_76423028: A / C 9_76451948: C / T 9_76542644: G / A 9_76588558: T / A 9_76620412: C / T 9_76702059: G / A 9_76728407: T / C 9_76790763: C / T 9_76822276: G / A 9_76923533: C / T 9_76968623: A / T 9_77014933: C / T 9_77020586: T / C 9_77023681: C / T 9_77036240: T / G 9_77037116: A / G 9_77037845: T / G 9_77042726: G / T 9_77045263: A / G 9_77046543: C / T 9_77051045: T / G 9_77051841: C / T 9_77052624: A / T 9_77052644: G / T 9_77053097: A / C 9_77053368: C / T 9_77054912: T / A 9_77056383: A / T 9_77056599: C / T 9_77112200: C / A 9_77117401: C / T 9_77120702: A / T 9_77123968: T / G 9_77143623: C / T 9_77145213: A / C 9_77151335: C / T 9_77158596: T / C 9_77158903: C / T 9_77159806: A / G 9_77222805: G / A 9_77289760: C / T 9_77327486: A / G 9_77365820: C / T 9_77465629: T / C 9_77499069: G / A 9_77564214: A / G 9_77625939: T / C 9_77689896: G / A 9_77729522: A / C 9_77799566: C / T 9_77837745: C / G 9_77879101: T / C 9_77881725: A / G 9_77890071: T / C 9_77895252: A / G 9_77908207: T / C 9_77915795: T / C 9_77928781: T / C 9_77931486: C / T 9_77999692: C / T 9_78027390: C / T 9_78027390: C / T 9_78112235: T / C 9_78150870: T / C 9_78218269: C / G 9_78249250: T / C 9_78343967: G / A 9-78405464: G / T 9_78455317: G / T 9_78497498: C / T 9_78568304: T / C 9_78620063: A / G 9_78672815: T / C 9_78710738: A / G 9_78760852: A / G 9_78801821: G / A 9_78872475: C / A 9_78939921: A / G 9_79007316: A / G 9_79059358: G / A 9_79104835: C / T 9_79173926: T / C 9_79268547: A / C 9_79308157: C / T 9_79341428: T / C 9_79440186: T / C 9_79448927: G / A 9_79522809: C / T 9_79587395: T / C 9_79616898: T / C 9_79683111: C / T 9_79774104: A / G 9_79818710: T / C 9_79843805: T / C 9_79890260: A / G 9_79961103: G / A 9_80028568: C / T 9_80099662: T / C 9_80130425: T / C 9_80213605: G / A 9_80232217: C / T 9_80320932: C / G 9_80345328: G / A 9_80410894: C / T 9_80460249: A / C 9_80539635: T / C 9_80557808: C / T 9_80637076: T / C 9_80703930: T / C 9_80725260: A / G 9_80783098: G / A 9_80857325: G / A 9_80918557: C / T 9_80938951: T / C 9_80984150: A / T 9_81076245: C / T 9_81122306: G / T 9_81179882: G / T 9_81235945: T / C 9_81296627: G / A 9_81345822: A / G 9_81413429: C / T 9_81434086: A / G 9_81513364: T / G 9_81536081: T / C 9_81619172: G / A 9_81682682: G / C 9_B1738994: C / T 9_81792744: G / A 9_81825895: T / C 9_81877636: C / T 9_81948375: A / C 9_81997991: T / G 9_82028823: A / C 9_82129402: C / A 9_82147943: T / C 9_82203546: C / G 9-82262945: C / T 9_82324994: T / C 9_82393878: T / C 9_82413574: C / T 9_82493338: A / G 9_82559599: A / T 9_82617399: A / C 9_82651946: A / G 9_82696189: C / T 9_82753473: C / T 9_82824124: G / A 9_82886654: C / T 9_82914218: C / T 9_82979980: C / T 9_83062465: A / G 9_83107380: C / T 9_83172484: G / A 9_83181702: C / T 9_83253835: G / A 9_83313636: T / A 9_83362080: A / G 9_83396954: A / G 9_83493723: G / A 9_83555602: A / G 9_83582369: A / G 9_83618040: C / T 9_83672735: A / T 9_83779870: A / G 9_83823806: C / T 9_83857242: G / A 9_83943647: A / T 9_83982910: A / G 9_84004408: T / C 9-84090416: T / C 9_84155002: C / T 9_84166135: G / A 9_84271203: T / C 9_84321463: G / A 9_84361811: G / A 9_84407625: C / T 9_84475372: C / T 9_84547024: C / T 9_84576699: A / G 9_84622783: C / A 9_84697952: C / A 9_84723342: G / A 9_84791380: G / A 9_84842120: C / A 9_84926498: A / G 9_84957481: C / A 9-85022053: G / A 9_85076328: C / T 9_85104024: T / C 9_85177456: A / G 9_85229796: G / T 9_85273885: A / G 9_85359052: G / A 9_85397557: G / A 9_85457821: A / G 9_85488722: A / G 9_85566081: T / A 9_85627892: G / A 9_85660444: A / G 9_85732391: A / G 9_85776233: G / A 9_85830856: C / T 9_85871086: A / G 9_85919849: T / C 9_85996614: G / A 9_86081365: T / G 9_86113612: A / / G 9_86164372: T / C 9_86222199: T / C 9_86290981: T / C 9_86318558: T / C 9_86361685: G / A 9_86466725: T / C 9_86486532: T / C 9_86571632: G / A 9_86587636: T / C 9_86632715: T / C 9_86697253: C / A 9_86742005: G / T 9_86825941: T / C 9_86858877: G / C 9_86957202: C / T 9_87009183: A / G 9_87058692: A / G 9_87111770: T / C 9_87158766: A / G 9_87211564: A / T 9_87263298: A / G 9_87291304: C / T 9_87366826: G / T 9_87453080: T / C 9_87492048: A / G 9_87553446: T / C 9_87613197: C / T 9_87621536: G / A 9_87685693: C / T 9_87747858: T / C 9_87802249: A / G 9_87878162: A / C 9_87923258: A / G 9_87981335: A / G 9_88010625: A / G 9_88062717: G / C 9_88135349: T / G 9_88214373: T / G 9_88246153: C / T 9_88309078: A / T 9_88331649: C / A 9_88398350: G / T 9_88470087: G / A 9_88498115: T / G 9_88573343: A / C 9_88649238: T / C 9_88702575: G / A 9_88757371: A / G 9_88815015: T / C 9_88837043: A / G 9_88898593: G / A 9_88940247: G / A 9_89037139: A / C 9_89083454: C / T 9_89134174: G / C 9_89201781: A / G 9_89225558: C / A 9_89280488: G / A 9_89360059: T / C 9_89422910: G / A 9_89469808: G / A 9_89509029: A / G 9_89591246: G / A 9_89623315: G / T 9_89662352: C / T 9_89751101: G / C 9_89786024: C / G 9_89829484: T / A 9_89912375: A / G 9_89955211: T / C 9_90007263: A / T

[0348] Table 102 continuation of Table 1

[0349] Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt Chr_Pos: Ref / Alt 9_90082862: A / G 9_90118189: T / C 9_90318133: A / G 9_90364600: G / T 9_90468458: A / G 9_90478525: G / A 9_90571353: C / G 9_90606511: A / G 9_90634963: T / A 9_90718602: G / A 9_90750469: C / T 9_90811464: C / A 9_90893222: C / T 9_90932320: A / G 9_90970708: C / A 9_91064793: G / A 9_91090578: G / A 9_91178187: A / C 9_91200268: G / C 9_91277480: T / C 9_91335084: C / T 9_91373904: T / C 9_91434241: A / G 9_91497137: A / G 9_91552033: G / T 9_91602419: A / G 9_91624669: A / G 9_91708193: A / C 9_91760017: A / G 9_91815982: C / G 9_91857699: T / C 9_91926097: A / G 9_91993619: A / C 9_92025468: G / A 9_92097975: G / A 9_92155417: C / T 9_92206871: C / T 9_92267404: A / G 9_92317660: T / C 9_92366642: T / G 9_92426368: A / G 9_92495600: C / T 9_92538870: T / A 9_92599403: G / A 9_92639194: C / T 9_92699811: A / G 9_92765794: T / C 9_92802800: C / T 9_92844172: C / T 9_92888561: C / A 9_92972930: A / G 9_93003514: T / G 9_93068658: G / T 9_93153984: C / T 9_93188905: A / C 9_93261224: C / T 9_93286548: G / A 9_93338194: T / C 9_93429156: G / C 9_93474392: C / T ​ 9_93523998: T / C 9_93548751: A / G 9_93632548: G / A 9_93652808: C / T 9_93756551: T / C 9_93809628: C / T 9_93831017: G / A 9_93914069: T / G 9_93964275: T / C 9_93996588: A / G 9_94079916: G / C 9_94134965: T / C 9_94186909: A / G 9_94209145: C / T 9_94305604: A / G 9_94348700: C / T 9_94410318: TT / C 9_94418528: G / C 9_94501264: C / T 9_94574193: A / G 9_94633030: A / G 9_94676540: A / G 9_94725060: C / G 9_94749284: G / A 9_94846602: C / T 9_94901698: C / G 9_94960522: A / G 9_94975199: C / T 9_95070097: T / C 9_95110872: A / G 9_95141834: A / T 10_93194: T / C 10_149789: C / T 10_200689: C / A 10_252985: A / G 10_319750: G / A 10_357520: A / C 10_437877: A / G 10_459077: G / A 10_510206: G / A 10_601755: G / C 10_634232: G / A 10_666304: G / A 10_761273: T / A 10_782128: A / G 10_864866: T / C 10_931141: G / A 10_988940: G / A 10-1044847: C / T 10_1144495: A / G 10_1198291: C / A 10_1253336: G / T 10_1290604: T / G 10_1363171: T / C 10-1408947: A / C 10-1471619: A / T 10_1488902: G / A 10_1564994: C / T 10_1627534: C / T 10_1685004: T / G 10_1751333: G / C 10_1808676: A / G 10_1834446: C / G 10_1892012: G / A 10_1963173: A / T 10-1985639: C / T 10_2068880: A / G 10_2135865: A / G 10_2181262: G / A 10_2205732: T / C 10_2268288: A / C 10_2341171: G / A 10_2369584: A / C 10_2426007: A / C 10-2485870: A / C 10_2575570: A / T 10_2589725: T / C 10-2686211: C / T 10_2726277: C / T 10_2773619: C / T 10_2832165: T / A 10_2875597: G / A 10_2936580: C / T 10_2964910: C / A 10_3057738: A / G 10_3076969: T / C 10_3137456: G / A 10_3205243: A / G 10_3246695: C / T 10_3358725: C / T 10_3449377: C / A 10_3499320: A / G 10_3528820: A / G 10_3589006: T / C 10_3664938: T / A 10_3682144: G / A 10_3765797: A / G 10_3821978: G / A 10_3838411: T / C 10_3933580: T / G 10_3961155: T / C 10_4051039: G / A 10_4110177: T / C 10_4155026: C / T 10_4156766: G / A 10_4159472: A / G 10_4161867: T / G 10_4164895: T / C 10__4165312: G / C 10_4165750: A / G 10_4170528: G / A 10_4170796: G / A 10__4173594: A / G 10_4174699: G / A 10)4175464: T / C 10_4181033：A℃ 10_4185352: C / T 10_4186402: A / C 10_4187037: A / G 10_4187161: A / C 10__4275301: G / A 10_4295254: A / G 10_4370060: C / T 10_4406746: G / A 10_4487939: G / A 10_4505227: A / G 10_4591131: C / A 10_4637896: G / T 10_4662844: A / T 10_4719126: T / C 10__4798545: C / A 10_4862012: C / T 10_4919011: C / T 10_4983327: G / A 10_5000779: G / A 10_5083270: C / T 10_5118908: A / T 10_5194773: G / / A 10_5262404: G / A 10_5303362: T / C 10_5353566: A / G 10_5395676: G / A 10_5453866: C / T 10_5495300: T / A 10_5554852: G / A 10_5645976: C / T 10_5682435: A / G 10_5753491: T / C 10_5772163: A / T 10_5862776: C / T 10_5883529: T / C 10_5959225: A / C 10_5982692: A / T 10_6047296: C / T 10_6104534: T / G 10_6185842: T / C 10_6220087: T / G 10_6298877: T / A 10_6349701: A / G 10_6413283: C / T 10_6435838: A / T 10_6505940: G / T 10_6537446: T / A 10_6589746: G / A 10_6735056: A / / G 10_6789821: C / A 10-6853271: G / A 10_6880194: A / G 10_6947849: A / C 10_6967952: t / C 10_7018338: C / T 10_7089618: A / T 10_7163913: T / C 10_7215650: G / T 10_7259919: G / A 10_7324000: T / C 10_7362335: A / C 10-7428645: T / C 10_7482121: T / C 10_7558834: A / C 10_7575549: T / C 10_7666791: A / C 10_7702933: G / C 10_7783486: C / T 10_7862280: G / T 10_7907572: A / C 10_7965217: A / G 10_8046929: C / T 10_8089963: G / T 10_8118239: T / C 10_8198920: C / T 10_8273132: G / T 10_8321650: C / T 10_8348082: G / A 10_8439182: A / T 10_8490032: G / A 10_8527495: T / A 10_8572236: C / T 10_8642518: G / A 10_8664987: G / C 10_8772665: T / C 10_8797446: T / C 10_8867717: G / A 10_8883664: A / G 10_8962065: C / G 10_9041318: C / T 10_9095834: A / G 10_9105428: C / A 10_9206512: G / A 10_9245791: A / G 10_9280693: A / T 10_9351298: C / T 10_9395091: T / A 10_9476958: G / T 10_9533058: A / G 10_9551882: G / T

[0350] Table 103 continuation of Table 1

[0351]

[0352]

[0353] Table 104 continuation of Table 103

[0354]

[0355]

[0356] Table 105 continuation of Table 104

[0357]

[0358]

[0359] Table 106 continuation of Table 105

[0360]

[0361]

[0362] Table 107 continuation of Table 106

[0363]

[0364]

[0365] Table 108 continuation of Table 107

[0366]

[0367]

[0368] Table 109 continuation of Table 108

[0369]

[0370]

[0371] Table 110 continuation of Table 109

[0372]

[0373]

[0374] Table 111 continuation of Table 110

[0375]

[0376]

[0377] Table 111 continuation of Table 1

[0378]

[0379]

[0380] Table 112 continuation of Table 1

[0381]

[0382]

[0383] Table 113 continuation of Table 1

[0384]

[0385]

[0386] Table 114 continuation of Table 1

[0387]

[0388]

[0389] Table 115 continuation of Table 1

[0390]

[0391]

[0392] Table 116 continuation of Table 1

[0393]

[0394]

[0395] Table 117 continuation of Table 1

[0396]

[0397]

[0398] Table 118 continuation of Table 1

[0399]

[0400]

[0401] Table 120 continuation of Table 1 19

[0402]

[0403]

[0404] Table 121 continuation of Table 120

[0405]

[0406]

[0407] Table 122 continuation of Table 121

[0408]

[0409]

[0410] Table 123 continuation of Table 122

[0411]

[0412]

[0413] Table 124 continuation of Table 123

[0414]

[0415]

[0416] Table 125 continuation of Table 124

[0417]

[0418]

[0419] Table 126 continuation of Table 125

[0420]

[0421]

[0422] Table 127 continuation of Table 126

[0423]

[0424]

[0425] Table 128 continuation of Table 127

[0426]

[0427]

[0428] Table 129 continuation of Table 1

[0429]

[0430]

[0431] Table 130 continuation of Table 1

[0432]

[0433]

[0434] Table 131 continuation of Table 1

[0435]

[0436]

[0437] Table 132 continuation of Table 1

[0438]

[0439]

[0440] Table 133 continuation of Table 1

[0441]

[0442]

[0443] Table 134 continuation of Table 1

[0444]

[0445]

[0446] Table 135 continuation of Table 1

[0447]

[0448]

[0449] Table 136 continuation of Table 1

[0450]

[0451]

[0452] Table 137 continuation of Table 1

[0453]

[0454]

[0455] Table 137 continuation of Table 1

[0456]

[0457]

[0458] Table 138 continuation of Table 1

[0459]

[0460]

[0461] Table 139 continuation of Table 1

[0462]

[0463]

[0464] Table 140 continuation of Table 1

[0465]

[0466]

[0467] Table 141 continuation of Table 1

[0468]

[0469]

[0470] Table 142 continuation of Table 1

[0471]

[0472]

[0473] Table 143 continuation of Table 1

[0474]

[0475]

[0476] Table 144 continuation of Table 1

[0477]

[0478]

[0479] Table 145 continuation of Table 1

[0480]

[0481]

[0482] Table 146 continuation of Table 1

[0483]

[0484]

[0485] Table 147 continuation of Table 1

[0486]

[0487]

[0488] Table 148 continuation of Table 1

[0489]

[0490]

[0491] Table 149 continuation of Table 1

[0492]

[0493]

[0494] Table 150 continuation of Table 1

[0495]

[0496]

[0497] Table 151 continuation of Table 1

[0498]

[0499]

[0500] Table 152 continuation of Table 1

[0501]

[0502]

[0503] Table 153 continuation of Table 1

[0504]

[0505]

[0506] Table 155 continuation of Table 1

[0507]

[0508]

[0509] Table 156 continuation of Table 1

[0510]

[0511]

[0512] Table 157 continuation of Table 1

[0513]

[0514]

[0515] Table 158 continuation of Table 1

[0516]

[0517]

[0518] Table 159 continuation of Table 1

[0519]

[0520]

[0521] Table 160 continuation of Table 1

[0522]

[0523]

[0524] Table 161 continuation of Table 1

[0525]

[0526]

[0527] Table 162 continuation of Table 1

[0528]

[0529]

[0530] Table 163 continuation of Table 1

[0531]

[0532]

[0533] Table 164 continuation of Table 1

[0534]

[0535]

[0536] Table 165 continuation of Table 1

[0537]

[0538]

[0539] Table 166 continuation of Table 1

[0540]

[0541]

[0542] Table 167 continuation of Table 1

[0543]

[0544]

[0545] Table 168 continuation of Table 1

[0546]

[0547]

[0548] Table 169 continuation of Table 1

[0549]

[0550]

[0551] Table 170 continuation of Table 1

[0552]

[0553]

[0554] Table 171 continuation of Table 1

[0555]

[0556]

[0557] Table 171 continuation of Table 1

[0558]

[0559]

[0560] Table 172 continuation of Table 1

[0561]

[0562]

[0563] Table 173 continuation of Table 1

[0564]

[0565]

[0566] Table 174 continuation of Table 1

[0567]

[0568]

[0569] Table 175 continuation of Table 1

[0570]

[0571]

[0572] Table 176 continuation of Table 1

[0573]

[0574]

[0575] Table 177 continuation of Table 1

[0576]

[0577]

[0578] Table 178 continuation of Table 1

[0579]

[0580]

[0581] Table 179 continuation of Table 1

[0582]

[0583]

[0584] Example 2: Application of the Tan Sheep SNP Liquid Chip in the Detection of Tan Sheep DNA Samples

[0585] 1. DNA extraction

[0586] The Tan sheep blood samples were subjected to DNA extraction using the magnetic bead method with the Kangwei Century CW2361S kit. The specific operation steps are as follows:

[0587] In a 2.0 mL lysis tube, add Tan sheep blood samples, Proteinase K and Buffer WL, and place in a vortex mixer for 30 s. Lysis in a 56℃ constant temperature water bath for 40 min, with shaking every 20 min. After lysis, centrifuge for a moment, and then add Lysate, Buffer KL and Mag beads PN solution in sequence. Insert the Spin tips Pack (magnetic rod sleeve) into the 96DW deep well plate, and run the DNA extraction program to complete the pDNA extraction and obtain the DNA sample.

[0588] The source of the Tan sheep blood samples was the Hong temple fortress district Tian Yuan good breed sheep breeding and breeding Co., Ltd., Wuzhong Hong temple fortress district Tian Yuan agricultural and pastoral technology development Co., Ltd. And Ningxia Yanchi Tan sheep industry group Shuo breeding Co., Ltd.

[0589] 2. DNA quality inspection

[0590] S1. Genome integrity detection: take 2 μL of DNA sample and spot on a 1% agarose gel, and run electrophoresis at 150 V for 25 min. Place the gel in a gel imager (Tanon Tanon1600). If there is only one band type, the DNA sample has good integrity.

[0591] S2. Purity detection: use NanoDrop 2000 nucleic acid protein detector to detect the purity of the DNA sample. If the A260 / A280 ratio is within the range of 1.7-2.1, and the A260 / A230 ratio is within the range of 1.8-2.2, the purity of the DNA sample meets the requirements of library construction.

[0592] S3. Quantitative detection: use the dsDNA HS Assay Kit for Qubit kit to accurately quantify the sample by taking 1 μL of DNA sample. The total amount should be >1 ug to meet the subsequent library construction.

[0593] 3. Targeted capture sequencing detection

[0594] 3.1, Genome DNA fragmentation

[0595] First, take 1.5 μg of DNA sample in a 0.6 mL tube, and add TE buffer to 100 μL, shake well and centrifuge. Second, set the fragmentation time of the fragmentation instrument to 30 s, the stop time to 30 s, and the number of fragmentation cycles to 6 cycles, and place the fragmentation tube symmetrically on the rotor of the fragmentation instrument to perform the fragmentation operation. Finally, after fragmentation, the fragment is detected, 10 μL of the fragmented sample is taken in a 96-well PCR, 3.5 μL of bromophenol yellow is mixed well by blowing, and is spotted on a 3% agarose gel, 170 V, 25 min electrophoresis detection is performed, and the main fragment size is required to be 200-300 bp, and the fragmentation product is obtained.

[0596] 3.2, Whole genome library construction

[0597] (1) Fragment screening

[0598] S1. Take 90 μL of the fragmentation product into a new labeled 96-well purification plate, then take 10 μL of sterile water into the 90 μL fragmentation product, and then take 85 μL of magnetic beads into the 100 μL fragmentation product, mix well by blowing 10 times with a pipette, and incubate at room temperature for 5 min.

[0599] S2. Place the 96-well purification plate on the magnetic stand, and let it stand for 5 min until the liquid is clear. Use a pipette to aspirate 180 μL of supernatant into a new 96-well purification plate.

[0600] S3. Take 20 μL of magnetic beads into the 180 μL supernatant, mix well by blowing 10 times with a pipette, and incubate at room temperature for 5 min.

[0601] S4. Place the 96-well purification plate on the magnetic stand, and let it stand for 5 min until the liquid is clear. Use a pipette to aspirate and discard the supernatant.

[0602] S5. Keep the 96-well purification plate fixed on the magnetic stand, add 200 μL of freshly prepared 80% (v / v) ethanol, and carefully discard the supernatant after standing at room temperature for 30 s.

[0603] S6. Repeat step S5 once, and try to aspirate the liquid at the bottom of the plate as much as possible. When there is a small amount of liquid residue, use a small volume pipette to aspirate the liquid at the bottom of the plate.

[0604] S7. Keep the 96-well purification plate fixed on the magnetic stand, and dry at room temperature until the magnetic beads have no reflection and no cracking.

[0605] S8. Take the 96-well purification plate off the magnetic stand, add 27 μL of sterile water to elute the DNA, mix well by blowing 10 times with a pipette, and dissolve at room temperature for 5 min.

[0606] S9. Place the 96-well purification plate on a magnetic stand and let it sit for 5 min until the liquid is clear. Transfer 25 μL of the supernatant to a new 96-well PCR plate for the next reaction.

[0607] (2) End Repair

[0608] Prepare the end repair reaction solution according to Table 181 on ice.

[0609] Table 181 Preparation of end repair reaction solution

[0610] Component Volume / μL Fragmented DNA / cfDNA 40 End Repair & A-Tailing Buffer 6 End Repair & A-Tailing Enzyme 4 Total 50

[0611] Using a pipette, add 10 μL of the end repair reaction solution to the 6-well PCR plate, seal the film, vortex to mix, and centrifuge briefly to collect the reaction solution at the bottom of the tube. Perform the reaction according to the reaction program in Table 182.

[0612] Table 182 Reaction program for end repair

[0613] Temperature Time Hot lid (70°C) On 20℃ 30 min 65℃ 30 min

[0614] After the reaction is completed, centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0615] (3) Adapter Ligation

[0616] In the 96-well PCR plate of step (2), add 5 μL of CAGT Universal Adapters, and using a pipette, add 20 μL of Ligation Master Mix to the 96-well PCR plate, seal the film, vortex to mix, and centrifuge briefly to collect the reaction solution at the bottom of the tube. Perform the reaction according to the reaction conditions in Table 183.

[0617] Table 183 Reaction conditions for adapter ligation

[0618] Temperature Time Hot lid (105°C) Off 20℃ 15 min 20℃ Hold

[0619] After the reaction is completed, centrifuge briefly to collect the reaction solution at the bottom of the tube, and transfer all the product to a new 96-well purification plate for purification operation to obtain the ligation product.

[0620] (4) Purification of Ligation Product

[0621] S1. Add 60 μL of magnetic beads to 75 μL of ligation product, and using a pipette, gently blow 10 times to mix thoroughly, and incubate at room temperature for 5 min.

[0622] S2. Place the 96-well purification plate on a magnetic stand and let it sit for 2-5 min until the liquid is clear. Carefully aspirate and discard the supernatant using a pipette.

[0623] S3. Keep the 96-well purification plate fixed on the magnetic stand, add 200 μL of freshly prepared 80% (v / v) ethanol, and carefully discard the supernatant after standing at room temperature for 30 s.

[0624] S4. Repeat the above step once, and try to absorb the liquid at the bottom of the plate as much as possible. When there is a small amount of liquid left, use a small volume pipette to absorb the liquid at the bottom of the plate.

[0625] S5. Keep the 96-well purification plate fixed on the magnetic stand, and dry at room temperature until the magnetic beads have no reflection and no cracking.

[0626] S6. Take the 96-well purification plate off the magnetic stand, add 17 μL of TE buffer for DNA elution, and mix well by gently pipetting 10 times with a pipette. Dissolve at room temperature for 5 min.

[0627] S7. Place the 96-well purification plate on the magnetic stand, and let it stand for 5 min until the liquid is clear. Transfer 15 μL of the supernatant to a new 96-well PCR plate.

[0628] (5) Pre-PCR amplification enrichment and purification

[0629] Take 15 μL of the ligation-purified product and prepare the reaction system on ice according to Table 184.

[0630] Table 184 Reaction system for Pre-PCR amplification enrichment and purification

[0631] Component Volume / μL CAGT UDI Primer 10 Equinox Library Amp Mix (2x) 25 Adapter Ligated DNA 15 Total 50

[0632] Use a pipette to take 35 μL of the prepared PCR amplification reaction liquid and add it to a 96-well PCR plate. Seal the film, vortex to mix, and centrifuge to collect the reaction liquid at the bottom of the tube. Perform PCR reaction according to the conditions in Table 185.

[0633] Table 185 PCR reaction program

[0634]

[0635] After the reaction is completed, centrifuge the reaction liquid to the bottom of the tube and perform product purification operation.

[0636] S1. Take 50 μL of magnetic beads into a 96-well purification plate containing 50 μL of PCR product, mix thoroughly by gently pipetting at least 10 times with a pipette, and incubate at room temperature for 5 min.

[0637] S2. Place the 96-well purification plate on the magnetic stand, and let it stand for 5 min until the liquid is clear. Carefully aspirate and discard the supernatant with a pipette.

[0638] S3. Keep the 96-well purification plate fixed on the magnetic stand, add 200 μL of freshly prepared 80% (v / v) ethanol, and carefully discard the supernatant after standing at room temperature for 30 s.

[0639] S4. Repeat the above step once, and try to absorb the liquid at the bottom of the plate as much as possible. When there is a small amount of liquid left, use a small volume pipette to absorb the liquid at the bottom of the tube.

[0640] S5. Keep the 96-well purification plate fixed on the magnetic stand, and dry at room temperature until the magnetic beads have no reflection and no cracking.

[0641] S6. Take the 96-well purification plate off the magnetic stand, add 22 μL of TE Buffer for DNA elution, and mix well by gently blowing the pipette for at least 10 times. Dissolve at room temperature for 5 min.

[0642] S7. Place the 96-well purification plate on the magnetic stand, and stand for 5 min until the liquid is clear. Transfer 20 μL of supernatant to a new 96-well plate.

[0643] 3.3, Whole genome library quality inspection

[0644] Use the dsDNA HS Assay Kit for Qubit kit to quantify the sample (1 μL sample + 199 μL Buffer), and take 80 ng of PCR purified product according to the concentration obtained by quantification in a 96-well PCR, add 3.5 μL of bromophenol yellow, mix well after blowing, and detect by electrophoresis in a 3% agarose gel at 170 V for 25 min. The qualified library fragment size is about 350-420 bp. Finally, the whole genome library construction is completed.

[0645] 4, Probe hybridization capture

[0646] 4.1, Library pool

[0647] The amount of DNA library is shown in Table 186. The total amount can be more than 1500 ng, but not more than 4 ug.

[0648] Table 186 DNA library usage

[0649] Mixed sample number Usage per library Total amount of each reaction library 1 500 ng 500 ng 2 500 ng 1500 ng 3 500 ng 1500 ng 4 375 ng 1500 ng 8 187.5 ng 1500 ng

[0650] Add the pre-hybridization reagent in Table 187 to the mixed sample, mix well, and try not to generate bubbles.

[0651] Table 187 Preparation of pre-hybridization reagent

[0652] Reaction Volume CAGT probe 4 μL Universal blocking reagent 8 μL Blocking reagent solution 5 μL

[0653] Dry the mixed pre-hybridization reagent above at room temperature in a vacuum concentrator, and perform the hybridization step.

[0654] 4.2, Probe hybridization

[0655] Incubate the Fast Hybridization Mix at 65°C for 10 min or until all the precipitate is dissolved, vortex quickly and add 20 μΐ, to the freeze-dried sample from the previous step to resuspend the sample (do not allow the hybridization solution to return to room temperature), flick the tube with your finger to mix, avoiding the formation of air bubbles, centrifuge briefly to remove the air bubbles, add 30 μΐ, of Hybridization Enhancer to the surface of the reagents, place the PCR tube in a pre-heated PCR machine for hybridization, see Table 188 for the program, and use a hot lid at 85°C.

[0656] Table 188 Probe hybridization reaction program

[0657] Temperature Time 95℃ Hold 95℃ 5 min 60℃ 15 min-4 h

[0658] 4.3, Post-hybridization elution

[0659] S1. Shake the pre-equilibrated streptavidin magnetic beads until they are well mixed, add 100 μΐ, of the magnetic beads to a 1.5 mL centrifuge tube, add 200 μΐ, of the binding buffer and mix well by flicking the tube with your finger.

[0660] S2. Place the centrifuge tube in a magnetic stand for 1 min or until the solution is clear, discard the supernatant and remove the tube from the stand.

[0661] S3. Repeat the washing step above 2 more times for a total of 3 times.

[0662] S4. After the final wash, add 200 μΐ, of the binding buffer and resuspend the beads by shaking until they are well mixed.

[0663] S5. After the hybridization is complete, open the lid of the PCR machine and quickly transfer all of the hybridization solution to the equilibrated magnetic beads.

[0664] S6. Mix the magnetic beads with the added hybridization solution well on a shaker, rocker or rotator at room temperature for 30 min.

[0665] S7. Remove the centrifuge tube from the shaker, centrifuge briefly and place the tube on the magnetic stand for 1 min, discard the supernatant and remove the tube from the stand; add 200 μΐ, of pre-warmed wash solution 1 and mix well; incubate at 70°C for 5 min.

[0666] S8. Place the centrifuge tube on the magnetic stand for 1 min, discard the supernatant and remove the tube from the stand; repeat the above step and add 200 μΐ, of pre-warmed wash solution 1 again, mix well; incubate at 70°C for 5 min.

[0667] S9. Transfer the liquid to a new tube; place on the magnetic stand for 1 min, remove the supernatant, and remove the tube; add 200 μL of pre-warmed wash buffer 2, mix by pipetting; incubate at 48°C for 5 min; place on the magnetic stand for 1 min, remove the supernatant, and remove the tube.

[0668] S10. Repeat the above steps for 2 more times, for a total of 3 times; for the last time, use 10 μL pipette tip to remove the wash buffer completely;

[0669] S11. Add 45 μL of water, mix, and incubate the solution on ice.

[0670] 4.4, Post-PCR enrichment, purification

[0671] Mix the magnetic bead mixture in Step 4.3, pipette out 22.5 μL to a 0.2 mL PCR tube, and add the PCR reaction system in Table 189.

[0672] Table 189 PCR reaction system

[0673] Reagent Volume / reaction Amplification primer 2.5 μL KAPA HiFi HotStart ReadyMix 25 μL Total 50 μL

[0674] Mix by pipetting, centrifuge quickly, and then place in a PCR machine to perform the reaction program in Table 190.

[0675] Table 190 PCR reaction program

[0676]

[0677] S1. Add 90 μL (1.8x) DNA purification magnetic beads to the amplified product, vortex to mix thoroughly; incubate at room temperature for 5 min; place the centrifuge tube on the magnetic stand for 1 min, and remove the supernatant after the solution is clear.

[0678] S2. Add 200 μL of freshly prepared 80% ethanol, incubate for 1 min, and discard the supernatant; repeat the 80% (v / v) ethanol wash once (total of 2 times), keep the centrifuge tube on the magnetic stand; carefully remove the residual ethanol with a 10 μL pipette tip, place at room temperature for 5-10 min or until the magnetic beads are dry, please note not to dry the magnetic beads too much.

[0679] S3. Remove the tube from the magnetic stand and add 32 μL of water, mix thoroughly by pipetting, and incubate at room temperature for 2 min; place the centrifuge tube on the magnetic stand for 3 min or until the solution is clear; transfer 30 μL of the supernatant to a clean 0.2 mL centrifuge tube.

[0680] 4.5, Quality inspection of capture library

[0681] The library was initially quantified using the dsDNA HS Assay Kit for Qubit kit (1 μL sample + 199 μL Buffer), and according to the quantification results, the effective concentration of the library was precisely quantified using qPCR; at the same time, the size of the library fragments was evaluated using the FA capillary electrophoresis system, and the qualified library fragment size should be between 350 bp and 450 bp.

[0682] 5. Genotype data analysis

[0683] After quality inspection, according to the effective concentration of the library and the demand for target data volume, pooling was performed, using Huada Zizao DNBSEQ-T7 sequencer, selecting PE150 sequencing strategy, performing on-machine sequencing, and the data was finally stored in FASTQ file format. The raw data FASTQ file was subjected to adapter removal, filtration and other quality control steps by Fastp software to obtain clean data, which was then aligned to the Tibetan sheep reference genome ARS-UI_Ramb_v3.0 by BWA-MEM software, and finally the HaplotypeCaller module in GATK software was used to generate SNP and INDEL variant site files, and the target sites were extracted, then 5x filtering was performed, and high-quality SNP typing of the target sites was obtained according to the indicators of DP greater than or equal to 5 and GQ greater than or equal to 20.

[0684] Example 3: Quality evaluation of genotype typing of Tibetan sheep 50K liquid chip obtained in Example 1

[0685] Randomly selected 105 Tibetan sheep individuals, extracted blood DNA, synthesized liquid chip probes according to the designed chip information, and detected 105 samples using liquid chip, and the results are shown in Table 191. As shown in Table 191, the sample site detection rate was between 98.8% and 99.42%, and the average detection rate was 99.23%.

[0686] Among them, the source of 105 Tibetan sheep individuals is Shuomumuhua Company of Yanchi Tibetan Sheep Industry Group in Ningxia.

[0687] Table 191 Detection rate of Tibetan sheep 50K SNP liquid chip

[0688]

[0689]

[0690]

[0691] Stability is generally measured by the consistency of genotype detection results of two replicate samples. Five samples were used to test the Tan sheep 50K SNP liquid phase chip in Example 1. The genotype detection results of the five replicate samples in two replicate samples are shown in Table 192. The genotype consistency was 99.7%, indicating that the genotype detection of Tan sheep 50K SNP liquid phase chip in Example 1 has good stability.

[0692] Table 192. Typing consistency of repeatable samples from Tan Sheep 50K SNP liquid phase chip.

[0693] Sample name Number of typing consistent sites Consistency rate of typing 25-344 49179 99.74％ 25-346 49189 99.78％ 25-343 49126 99.70％ 25-350 49160 99.68％ 25-345 49105 99.72％

[0694] The experimental results above show that the genotyping quality and stability of the Tan sheep 50K liquid phase chip are very good.

[0695] Example 4: Application of 50K liquid phase chip in whole-genome selection of Tan sheep

[0696] This invention uses accuracy, unbiasedness, and correlation as indicators for model evaluation. The accuracy of breeding values ​​is evaluated by using theoretical accuracy to assess the accuracy of different models. The theoretical accuracy of breeding value estimation is calculated using the following formula:

[0697]

[0698] In the above formula, r i To estimate the theoretical accuracy of breeding values; i = 1, 2, 3, ... are individual Tan sheep, SEP i It is the standard error of individual i's estimation of the breeding value; It is additive genetic variance.

[0699] The Pearson correlation coefficient between breeding values ​​and phenotypic values ​​is calculated using the following formula:

[0700]

[0701] In the above formula, r is the Pearson correlation coefficient between breeding value and phenotypic value; cov(a,p) is the covariance between the estimated breeding value and phenotypic value; var(a) and var(p) are the variances of the breeding value and phenotypic value, respectively.

[0702] The results show that the theoretical GEBV accuracy of the 15 traits calculated using the Tan Sheep 50K liquid phase chip SNP set is between 0.04 and 0.75, as shown in Table 193.

[0703] Table 193 GEBV Estimation Accuracy

[0704] Character Theoretical accuracy Pearson correlation coefficient BH 0.699 ± 0.021 0.892 BL 0.040 ± 0.053 0.710 MGJ 0.585 ± 0.030 0.802 BT 0.088 ± 0.088 0.662 BW 0.703 ± 0.020 0.802 CC 0.663±0.023 0.745 CD 0.623 ± 0.027 0.748 CW 0.660±0.024 0.790 GW 0.664 ± 0.023 0.830 MGG 0.671±0.023 0.862 SZBG 0.746±0.017 0.909 TC 0.637±0.026 0.817 TL 0.699 ± 0.021 0.877 TW 0.552±0.033 0.748 HS 0.450 ± 0.043 0.742

[0705] The results show that on the basis of the designed chip, combined with targeted capture sequencing (more SNPs than marker sites can be obtained without filling), the whole genome selection work can be carried out. Compared with the solid-phase chip of Tan sheep, the Tan sheep breeding work can be carried out at a lower cost.

[0706] It should be noted that when the numerical range is involved in the present application, it should be understood that each numerical range of two endpoints and any numerical value between the two endpoints can be selected. In order to prevent repetition, the present application describes the preferred embodiments.

[0707] Although the preferred embodiments of the present application have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept, and these changes and modifications all fall within the scope of all changes and modifications of the present application.

Claims

1. A liquid phase chip for Tan sheep 50K based on targeted capture sequencing, characterized in that, The Tan sheep 50K liquid phase chip consists of individually packaged probe mixtures and hybridization capture reagents; The individually packaged probe mixture includes probes for detecting 50,000 SNP sites associated with the economic traits of Tan sheep; the 50,000 SNP sites associated with the economic traits of Tan sheep are used together to design the probes, placing the target site in the middle of the probes. The 50,000 SNP loci associated with the economic traits of Tan sheep include 5,540 SNP loci on chromosome 1, 5,221 SNP loci on chromosome 2, 4,701 SNP loci on chromosome 3, 2,387 SNP loci on chromosome 4, 2,094 SNP loci on chromosome 5, 2,631 SNP loci on chromosome 6, 1,953 SNP loci on chromosome 7, 1,909 SNP loci on chromosome 8, 1,831 SNP loci on chromosome 9, 1,672 SNP loci on chromosome 10, 1,245 SNP loci on chromosome 11, 1,566 SNP loci on chromosome 12, and 1,566 SNP loci on chromosome 13. The chromosomes have 1759 SNP loci, chromosome 14 has 1307 SNP loci, chromosome 15 has 1655 SNP loci, chromosome 16 has 1481 SNP loci, chromosome 17 has 1487 SNP loci, chromosome 18 has 1316 SNP loci, chromosome 19 has 1284 SNP loci, chromosome 20 has 1068 SNP loci, chromosome 21 has 944 SNP loci, chromosome 22 has 977 SNP loci, chromosome 23 has 1204 SNP loci, chromosome 24 has 863 SNP loci, chromosome 25 has 1030 SNP loci, and chromosome 26 has 875 SNP loci.

2. The Tan Sheep 50K liquid phase chip according to claim 1, characterized in that, The probe has a GC content of 20% to 80%, a length of 120 bp, and a DNA nucleotide sequence modified with a biotin group at its 5' end.

3. The application of the Tan sheep 50K liquid phase chip as described in claim 1 in the analysis of important economic traits of Tan sheep.

4. The application according to claim 3, characterized in that, The important economic traits of Tan sheep are selected from at least one of the following: body length, body height, chest depth, chest width, chest circumference, cannon circumference, weight, cross height, backfat thickness, number of hair strands, number of shoulder strands, spike type, tail length, tail width, tail circumference, and tail type.

5. The application of the Tan sheep 50K liquid phase chip as described in claim 1 in Tan sheep genome selection and breeding.

6. The Tan sheep 50K liquid phase chip of claim 1 can be used in any one of the following applications: Tan sheep genetic diversity analysis, Tan sheep kinship identification, Tan sheep breed identification, genome-wide association analysis, and SNP genotyping detection.

7. The application according to any one of claims 5 or 6, characterized in that, The method for using the Tan sheep 50K liquid phase chip for SNP genotyping detection is as follows: Genomic DNA was extracted from the Tan sheep to be tested; a high-throughput sequencing library of Tan sheep DNA was constructed using the genomic DNA as a template. The Tan sheep DNA high-throughput sequencing library was hybridized and captured using the Tan sheep 50K liquid phase chip to enrich DNA fragments containing target SNP sites. The enriched DNA fragments were amplified and purified to obtain amplification products; The amplified products were subjected to high-throughput sequencing, and the sequencing results were compared with the sheep reference genome ARS-UI_Ramb_v3.

0. Use the HaplotypeCaller module of GATK software to generate SNP and InDel variant site files and extract target SNP sites. A 5× filter was used, combined with sequencing depth ≥ 5 and quality value GQ ≥ 20, to obtain high-quality SNP genotyping results for the target SNP loci.

8. The application according to claim 7, characterized in that, High-throughput sequencing was performed using the BGI DNBSEQ-T7 sequencing platform.

9. The application according to claim 7, characterized in that, The target SNP loci include functional or representative loci related to important economic traits, genetic diversity, kinship identification, or breed identification of Tan sheep.

10. The application according to claim 7, characterized in that, The SNP genotyping results of the high-quality target SNP loci are used for genetic evaluation, breed identification, kinship identification, or genome selection and breeding of Tan sheep.

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