- A new mouse SNP genotyping assay for speed congenics: combining flexibility, affordability, and power. - Results: We took advantage of the power of high throughput sequencing technologies to develop a cost-effective, high-density SNP genotyping assay that can be used across many combinations of backcross strains. - We demonstrated that the assay has a high density of diagnostic SNPs for backcrossing the BALB/c strain into the C57BL/6J strain SNPs), and a sufficient density of diagnostic SNPs for backcrossing the closely related substrains C57BL/6N and C57BL/6J SNPs).. - Furthermore, the assay can easily be modified to include additional diagnostic SNPs for backcrossing other closely related substrains. - We also developed a bioinformatic pipeline for SNP genotyping and calculating the percentage of alleles that match the backcross recipient strain for each sample. - We demonstrated the effectiveness of the assay and bioinformatic pipeline with a backcross experiment of BALB/c-IL4/. - IL13 into C57BL/6J. - Full list of author information is available at the end of the article. - The development of methods to create “ congenic ” mice has led to substantial advances in our understanding of the functions of genes and mutations (e.g. - Traditionally, the devel- opment of a congenic background has been accom- plished by backcrossing a mutant line with a standard inbred laboratory strain of the preferred genetic back- ground. - Although popularity has grown for new genome editing techniques that transfer genetic content to a new background without the need for backcrossing, such as the Cas9 based strategies, these techniques have disad- vantages compared to traditional approaches, including off-target effects and limitations in the length of the mutation that can be transferred [3 – 5]. - A major disad- vantage of the traditional congenic approach, however, is the length of time required for backcrossing. - These assays re- quire a separate set of diagnostic SNPs for each unique combination of backcross strains. - Other SNP arrays have been developed to survey genetic variation across mul- tiple strains and substrains using many thousands of SNPs (e.g., the Mouse Diversity Array [10] and the Mouse Universal Genotyping Arrays or MUGAs [9, 11]).. - the sequence data, including SNP genotyping and calculation of the percentage of alleles that match the backcross recipient strain for each sample. - We tested the performance of the assay on three commonly used backcross strains or substrains from multiple sources, and found the assay to have a high density of genome- wide SNPs for distinguishing strains BALB/c and C57BL/6J (807–819 SNPs) and a sufficient density of SNPs for distinguishing the closely related substrains C57BL/6J and C57BL/6N (123–139 SNPs). - We used prior published studies to identify SNPs for our genotyping assay that would be informative for speed congenics across a wide range of mouse strain combina- tions. - Probes for the target SNPs were 40 bp long and were custom-designed by Tecan using the UCSC mm10 genome assembly of the C57BL/6J strain (Acces- sion ID GCA as a reference. - Two probes were designed per target SNP, with one probe hybridiz- ing to the plus strand and the other to the minus strand, and each probe hybridizing within 100 bp of the target SNP. - For a small number of our target SNPs, probes could not be designed based on the criteria required by Tecan, or initial runs of the genotyping assay resulted in low numbers of sequence reads across samples. - design window by 60 bp on each side of the target SNP, and these new probes were added into the panel. - Y chromosome SNPs are not typically used for guiding speed congenics experiments, since the majority of the Y chromosome does not recombine and, there- fore, ancestry will be known based on the breeding strat- egy. - All procedures were approved by the Institutional Animal Care and Use Committee of the University of Idaho (protocol #IACUC-2020-10). - making for speed congenics experiments (Fig. - HTStream/releases/tag/v1.1.0-release) to remove PCR duplicates and adapter sequence, trim probe sequence (i.e., the first 40 bp of each forward read), and remove reads shorter than 90 bp. - Cleaned sequence reads are mapped to the reference genome of the backcross recipient strain using BWA v and mapping rates across samples are evaluated using MultiQC. - To assess sequencing perform- ance across SNPs for each sample, the number of mapped sequencing reads per sample and SNP are. - calculated using SAMtools v1.5 [18] with a bed file con- taining the reference genome locations of the target SNPs, and boxplots are created showing the distribution of the number of mapped sequence reads per SNP for each sample using R v3.6.0 [19].. - The pipeline outputs the SNP genotype calls for each sample, as well as a summary of the total percentage of alleles that match the reference allele for the 1640 auto- somal and X chromosome SNPs for each sample, and the number and percentage of SNPs with each possible genotype (homozygous for the reference allele, homozy- gous for the alternate allele, or heterozygous) for each sample. - The pipeline also outputs the percentage of SNPs that were successfully genotyped for each sample, to allow easy identification of samples that performed poorly.. - homozygous for different alleles between the two strains, and the evenness of the spacing of those SNPs across the genome. - To evaluate the effectiveness of our SNP panel for speed congenics for different combinations of strains and substrains, we determined the number and genomic distribution of diagnostic SNPs for backcrosses between two genetically divergent strains (donor BALB/. - To identify diagnostic SNPs for each donor strain (assuming the recipient strain is al- ways C57BL/6J), we conducted filtering steps to retain SNPs that consistently genotyped for the donor strain and were homozygous for a different allele than C57BL/. - We first filtered the SNP panel to remove SNPs that failed to genotype in more than one individual from the donor strain, and then removed SNPs for which any in- dividual from the donor strain was heterozygous or homozygous for the C57BL/6J allele. - To examine the spacing across the genome of the diagnostic SNPs for each donor strain, we calculated the number of SNPs per chromosome and the distance between adjacent SNPs on each chromosome. - for each set of diagnostic SNPs. - We further evaluated the effectiveness of the genotyping assay for speed congenics by using the assay to inform a backcross experiment with one of the donor strains (BALB/c-IL4/IL13, The Jackson Laboratory. - Table 1) into C57BL/6J. - We initially bred one male of the donor strain with two females of the recipient strain, and three male offspring from this cross were each bred with two females from the recipient strain. - We then conducted the genotyp- ing assay for all offspring of both sexes that had the gene of interest, using the bioinformatic pipeline to calculate the percentage congenic alleles across the diagnostic SNPs for each individual. - We used two to three breeders per generation and performed backcrosses until 99.8% of the congenic strain was achieved in the offspring, follow- ing standard congenics practices (e.g. - We chose to genotype all offspring containing the gene of interest at each generation to maximize the effectiveness of the speed congenics approach and thereby minimize the total number of generations required (Table S3) [23].. - We also performed bioinformatic analyses to predict the number of diagnostic SNPs for crosses of additional laboratory mouse strains. - We calculated the num- ber of predicted diagnostic SNPs for each cross as the number of SNPs with different genotypes between each pair of strains using R v3.6.0.. - 99.5% of reads mapping to the reference genome for each sample. - Assay performance for speed congenics. - After performing filtering steps to identify diagnostic SNPs for each donor strain (assuming the recipient strain is C57BL/6J), we identified 807 diagnostic SNPs for BALB/c-AnNHsd, 819 for BALB/c-IL4/IL13, 139 for C57BL/6N-Crl, and 123 for C57BL/6N-Hsd (Table 3).. - These diagnostic SNPs were distributed across all chro- mosomes for each donor strain. - For the backcross experiment of BALB/c-IL4/IL13 into C57BL/6J, the percentage of congenic alleles for the 819 diagnostic SNPs increased from a mean of 73.6% (range in the second backcross to a mean of 99.4%. - Bioinformatic analyses indicated the mean predicted number of diagnostic SNPs for crosses between each pair of 102 laboratory mouse strains was 549 ± 136 SD, with 95.2% of strain combinations having >. - 300 diagnostic SNPs (Table S4). - Table 2 Total number of demultiplexed sequence reads across three batches of 48 samples, and the mean and standard deviation of the number of sequence reads across samples within each batch. - because our assay includes a smaller number of SNPs (i.e., our assay uses 1499 of the 1638 SNPs reported in [13]).. - Furthermore, the assay is predicted to have a high density of diagnostic SNPs for many additional laboratory mouse strains, with a mean of 549 ± 136 SD. - diagnostic SNPs for crosses between 102 inbred and wild-derived inbred strains, and with 95.2% of strain combinations having >. - 300 diagnostic SNPs. - There- fore, our genotyping assay should be highly flexible for a wide variety of backcross strain combinations, and should have a high level of accuracy for characterizing the proportion of the genome that matches the recipient strain. - We also demonstrated that our assay has a suffi- cient density of genome-wide diagnostic SNPs for back- crossing the closely related substrains C57BL/6N and C57BL/6J, which are commonly used in congenics ex- periments (123–139 SNPs). - 2 Distributions of the numbers of sequence reads per SNP per sample for each of three batches of 48 samples. - Table 3 The number and chromosomal distribution of diagnostic SNPs for backcrosses from four donor strains into C57BL/6J. - Donor strain Diagnostic SNPs Number SNPs per chromosome Distance between adjacent SNPs (Mb). - Another advantage of our assay compared to commer- cial assays is that it allows the identification of a set of diagnostic SNPs for a backcross experiment without mak- ing any prior assumptions about the genomic composition of the original parental strains. - In contrast, analysis of the original parental strains with our genotyping assay would allow identification of a set of diagnostic SNPs tailored to a backcross experiment using those parental strains.. - 3 The chromosomal positions in the mouse genome of diagnostic SNPs for backcrosses into C57BL/6J from the following donor strains: a BALB/c-AnNHsd: 807 SNPs (b) BALB/c-IL4/IL13: 819 SNPs (c) C57BL/6N-Crl: 139 SNPs (d) C57BL/6N-Hsd: 123 SNPs. - calculation of the percentage of alleles matching the re- cipient strain for each sample. - The results of these ana- lyses are used to output a report of the genotypes for each sample and SNP, genotyping success rate across SNPs for each sample, and the number and percentage of alleles that match the recipient strain for each sample.. - This output allows easy identification and removal of samples with low genotyping performance, selection of the backcross offspring with the greatest percentage an- cestry from the recipient strain, and assessment of. - A description of the bio- informatic pipeline is available at https://github.com/. - kimandrews/CongenicMouseGenotyping, including step- by-step commands, scripts, a bed file of the target SNPs (this is also in Table S1), and a list of required dependencies.. - 4 Chromosomal distribution of diagnostic SNPs for backcrosses from donor strains (shown on the x-axis) into C57BL/6J, illustrated by the distribution of distances (number of base pairs) between adjacent SNPs that are divergent between C57BL/6J and the donor strain. - n % C57BL/6J alleles. - We developed a SNP genotyping assay for speed con- genics that takes advantage of the power of high through- put sequencing technologies to improve efficiency and affordability. - Standard-format “ bed ” file showing the genomic positions of the SNPs in the mouse genotyping assay. - First col- umn = chromosome number, second column = start of the SNP position, third column = end of the SNP position, fourth column = SNP name.. - Standard-format “ bed ” file showing the genomic positions of the probes in the mouse genotyping assay. - First column = chromosome number, second column = start of the SNP position, third column = end of the SNP position, fourth column = SNP name, fifth column = score, sixth column = positive or negative DNA strand. - the experiment identifying diagnostic SNPs for different strains. - name of the sequencing library. - number of SNPs that were homozygous for the alter- nate allele. - number of SNPs that were homozygous for the reference allele. - number of SNPs that failed to genotype. - total number of SNPs successfully genotyped. - Predicted numbers of diagnostic SNPs in the mouse genotyping assay for crosses between each pair of 102 inbred and wild-derived inbred mouse strains. - We thank Barrie Robison, Director of the University of Idaho IBEST, for supporting this work. - We thank Tecan for support with design of the genotyping assay, and two anonymous reviewers for helpful comments on the manuscript.. - KRA led the writing of the manuscript, KRA and SL edited the manuscript and all authors approved the manuscript. - Genomic positions of the SNPs and probes in the genotyping assay are provided as standard- format “ bed ” files in Tables S1 and S2. - A description of the bioinformatic pipeline is available at https://github.com/kimandrews/CongenicMouseGenotyping, including step-by- step commands, scripts, the bed file of the target SNPs (i.e., the same file as Table S1), and a list of required dependencies.. - All procedures used in this study were approved by the Institutional Animal Care and Use Committee of the University of Idaho (protocol #IACUC-2020-10).. - https://doi.org/10.1534/. - https://doi.org/10.1017/s . - https://doi.org . - https://doi.org/10.1534/g . - Content and performance of the MiniMUGA genotyping Array: a new tool to improve rigor and reproducibility in mouse research. - https://doi.org/10.1186/s . - https://doi.org/10.1093/. - https://doi.org/10.1016/s . - https://doi.org/10.1371/. - https://doi.org/10.1007/s y.. - On the subspecific origin of the laboratory mouse
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