FamPipe
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1. Freq score threshold file Compound heterozygosity Disease_model 2 Disease model type 1 Vi d usa ior a a Ds Me Mud allele transmissions in Families Folles a a patterns opa dedecus de d er re D3 Fhe disease medels are determined bsy the Follewine rules ant medel EFer an affeeted individual the genotype atthis SNP sheuld be DB or Dd Eoer an unatffeeted individual the genotype at4his SNP sheuld be dd QU EU MEM QE Geese sels de i ANM DE 24 Note if you only have a few pedierees please eonsider using external allele frequeney Elle 0 2 by speeifyine Mhe Freg hile se that Dandd ean be properly determined Segregation scores The segregation scores are calculated based on the algorithm in MendelScan http gmt genome wustl edu mendelscan Assuming a dominant model for a variant an individual is scored as follows Affected with dd 0 5 Affected with DD 0 8 Unaffected with Dd 0 1 Other 1 A dominant segregation score is calculated by multiplying the scores for all individuals for the variant Assuming a recessive model for a variant an individual is scored as follows Affected with dd 0 1 Affected with Dd 0 5 Unaffected with DD 0 1 Other 1 A recessive segregation score is calculated by multip2. frequency file is the same as the PLINK frequency file which contains six columns CHR Chromosome SNP SNP identifier Al Allele 1 code minor allele A2 Allele 2 code major allele MAF Minor allele frequency NCHROBS Non missing allele count Please see http pngu mgh harvard edu purcell plink thresh shtml maf The external frequency file can be either obtained automatically from our ftp site or provided by the user When connecting to our ftp site a list of frequency files for the 6 super populations i e AFR freq AMR freq EAS freq EUR freq and SAS freq estimated from the 1000 Genomes Project Data will be shown Please select an appropriate frequency file for your sample Command line options Download the external frequency file from our ftp site Freq file ftp Provide your own file Freq file file name Ex Freq file my freq Note Because the frequency files are large downloading the files from our ftp site for each analysis can be time consuming Alternatively these files are packaged into a single file and can be downloaded directly from the FamPipe website Please 5 download the file and use Freq file file name to avoid the download for each analysis The Cluster File Optional A cluster file which contains haplotype frequencies is used in Merlin to account for linkage disequilibrium LD For IBD and linkage analyses using Merlin a cluster file is not required in FamPipe as the
3. refer to the technical note Command line options Torun the IBD sharing analysis use this option BD Sharing Related options IBD threshold for P IBD The default value is 0 5 You can specify a higher threshold to get higher specificity i e more non IBD regions will be filtered However this may sacrifice the sensitivity 1 e may miss more IBD regions BD threshold value Example BD threshold 0 8 Tofilter IBD regions based on a threshold the Sharing threshold can be specified The default value is 0 5 Positions with the proportions of IBD pairs in all possible pairs of affected individuals gt Sharing threshold will be reported Sharing threshold value Example Sharing threshold 0 5 Grid of map positions for calculating the IBD probabilities The default value is 1 Please refer to the Merlin user manual for more information of the grid option grid value Example grid 0 5 13 PLINK prune threshold variance inflation factor VIF FamPipe uses PLINK to prune SNPs in LD using the VIF SNPs that are not correlated are used for the IBD analysis The default value of VIF is 2 Please see the PLINK manual Linkage disequilibrium based SNP pruning for more information vif value Example vif 1 5 IBD Sharing Output merlin s15 Output file for the Extended IBD state information from Merlin Extended IBD states track sharing of maternal and paternal allele
4. rule 4 A gene has two or more heterozygous variants in at least compound_rule4 100 of the affected individuals 5 In an affected child there is at least one variant transmitted from the paternal side and one transmitted from the maternal side At least compound ruleSs 10090 of the affected children follow the rule Note that when all compound rule thresholds are set to 1 the rules are the same as the rules described in Kamphans et al PLOS ONE 2013 Related options compound rulel value Default value is 1 compound rule2 value Default value is 1 compound rule3 value Default value is 1 compound_rule4 value Default value is 1 compound rule5 value Default value is 1 28 Output There are two columns in the output of compound heterozygosity the first column is the name of gene the second column is the count of SNPs in the gene which match the five rule For example compound heterozygosity result chrl txt Gene name of SNPs TBXI5 5 Other options verbose By default FamPipe does not output the screen output for the external programs to keep the screen clean Use this option to check the running details for the external programs 29 The Four Commonly Used Scenarios The four commonly used scenarios are discussed in the FamPipe paper We described the workflows for each of the scenarios Scenario 1 Small sample size filtering based approach PLINK files gt Calculate all
5. running FamPipe on UNIX we highly recommend the user especially the first time user to generate the command using the web based interface http X XX FamPipe can be launched with a command line like the following with many options for different analyses Jfampipe BED file name BIM file name FAM file name For example fampipe BED test bed BIM test bim FAM test fam Impute 1 Input files Output folder The PLINK BED BIM and FAM Files The PLINK bed bim and fam files are the three mandatory files to run FamPipe The format of the files can be found in the PLINK user manual http pngu mgh harvard edu purcell plink binary shtml The bed file is a binary file containing the genotype information The bim file contains the SNP names and map positions Chromosome number SNP names i e rs ID and physical positions based on hg19 should be specified The fam file contains the family structure where the affection status for each individual in the 6 column should be specified If your have both GWAS and NGS data they should be merged into a single set of the bed bim and fam files before running FamPipe Command line options Read the bed file BED file name Example BED example fampipe bed Read the BIM file BIM file name Example BIM example fampipe bim Read the FAM file FAM file name Example FAM example fampipe fam The External Program Path File FamPipe inte
6. 2 Imputation 0 No execute imputation Imputation 1 Merlin Imputation Imputation 2 GIGI Imputation interval File name must follow this regulation range chrX txt Association 0 1 2 Association 0 No execute association test Association 1 use Ordered subset Variable threshold Pedigree Disequilibrium Test OVPDT Association 2 use Family based Association Test FBAT Disease model 0 1 2 Disease model 0 not use Disease model type 1 function Disease model 1 use Disease model type 1 function Disease model 2 use compound heterozygosity function 33 Threshold option default Description Linkage pvalue 0 05 if p value 0 05 the grid is reported IBD threshold 0 5 If IBD HJf5 0 5 the pair of individuals is defined as IBD sharing grid 1 The gap of position in the output file of Linkage and IBD Analysis plink vif 2 plink indep 50 5 2 Sharing_threshold 0 5 If the number of pairs with IBD sharing number of pairs gt Sharing_threshold the grid is reported Disease model thresheld 08 The percentage of people who matches the rule for each marker has to be greater than this threshold Missing tolerance threshold cu An OS eerie Eee ee bee ee doe threshold compound rulel 0 1 compound_rule2 0 5 compound rul
7. FamPipe Alpha 1 1 Lastest update May 20 2015 User manual Content deed P H 1 Run the Program ccccccccccccecssssssnececececessesesaeeececeescsesesaeaeeeeeeecesenseaseseseeeesseesesaeaeeeeeeseneees 2 Input files Output TONE sceau ea rupti UNI dde QN tent MEN ene Un 3 The PLINK BED BIM and FAM Files s visitent dn aeo eco iiw Dod pep ERR Ple ue Use oup rule UR 3 The External Program Path File ccccssssccccccecssssssseseceesesesesseaeeeeeeeesseeseseaeeeeees 4 The External Frequency File Optional erepta sms 5 The Cluster File Optional csse ves tbc rk Eb XR ERU RR ERUEE camera RR rug Urea raa ts 6 The Interval File Optional ua rou icm mode pk uel d e ove RUD 7 The GWAS and NGS SNP List Files Required for running GIGI 8 The Annotation File Required for Association Tests 9 Did v qedrie c 10 FUNCTIONS sespcceceteicaccunpecsota tens a E a E eaaa 11 Update Genetic POSITIONS assii e E E E 11 ES UII NP MCN eM Pc a pco NE 12 IBD Sharing Analysis cccccsessscececececeessnseaeeeceeecessesneaeeeceeeesseesssasaeeeeeeseseseseaaees 13 LIN KAR SAMA YSIS cietat 16 Imputation Analysis ccccccesesscececeeeceesssseseeeeeeecessesseaeeeceeeessesseeaeeeeeesessseseeaeas 18 Merlin Imputation eeseeseseseseeeeeeeeenennnen nennen nnne nennen nnn 18 GIGI imputatilOrnoc sioe t i
8. e3 0 5 compound_rule4 0 8 compound_rule5 1 smallSwap 0 verbose 0 plink path merlin path pedcut path Freq_score 34 Error Warning Error Warning Description Error The genetic position in the third column in the bim file are duplicated Error The genetic position in the third column in the bim file is not correct value 500 Rutger Map chrl txt Error could not open Rutger Map file Error Can t find more than one SNP in the bim file that has identical name in Rutger s Map Warning could not find the result in linkage Analysis Warning There is no pvalue smaller than Linkage pvalue threshold in CHRI linkage txt that will not generate interval file Warning could not find the result in IBD Sharing Analysis Warning There is no IBD rate larger than Sharing threshold threshold in notable grid chrl that will not generate interval file Error could not find interval file Cannot find any interval for GIGI imputation please see Interval file Error could not open file Annotation file Annotation chrl txt Error could not find file gwas snpsl txt file Error could not find file NGS 1 Ist file 35 Citation Please cite the FamPipe paper Kang CY Yao PJ Tsai WY Chung RH 2015 FamPipe An automatic analysis pipeline for analyzing sequencing data i
9. ed for Association Tests Gene based association tests are performed using FBAT and OVPDT Therefore an annotation file is required to define gene regions Moreover IBD sharing statistics and disease model status will be added to the annotation file if the file is provided The file must have at least three essential columns starting with the column names chromosome position geneList For example in annotation txt chromosome position geneList 1 7324503 CAMTAI 1 7324547 CAMTAI 1 7324959 CAMTAI 1 7326621 CAMTAI 1 7326747 CAMTAI 1 7331060 CAMTAI Extra columns can be added in the file The annotation file should be based on NCBI 37 hg19 Command line options Anno_file filename Ex Anno_file annotation txt The Folder of Output User can assign the name of data output folder If user doesn t specify the output folder FamPipe will create a folder with the name famepipe date time FamPipe will also create a folder for each chromosome dir 1l dir 2 dir 3 under the data output folder Command line options out folder name For example out test FamPipe will create a folder named as test If the input file contains information of chromosomes 1 and 3 you will see the dir 1 folder and dir 3 folder in the test folder and analysis results for each chromosome are saved in its own folder 10 Functions Update Genetic Positions Programs such as Merlin and GIGI use the Haldane s map functions for
10. ele frequency gt Generate MERLIN map file gt Check large pedigree gt PedCut gt Split by chrom gt Prune for LD gt IBD sharing analysis gt Disease model gt Filtering gt Write results Command line J fampipe BED test bed BIM test bim FAM test fam IBD Sharing Disease model 1 Anno file annotation txt Output For example Association IBD chrl txt This output file contains all columns in the annotation file and additional four columns which are IBD rate IBD count IBD num of pair and Disease model Scenario 2 Large sample size association test in linkage regions PLINK files gt Calculate allele frequency gt Generate MERLIN map file gt Check large pedigree gt PedCut gt Split by chrom gt Prune for LD gt Linkage analysis gt Generate intervals gt Generate cluster file gt Imputation gt Merge the imputed file gt Run association test gt Write results Command line J fampipe BED test bed BIM test bim FAM test fam Linkage npl Association 1 Impute 1 Anno_file annotation txt Output For example Association linkage chrl txt Gene name of SNPs p value Linkage LOD TBXIS 354 0 516 0 01 This output contains four columns the first column is the name of genes the second column is the number of SNPs in genes the third columns is the association p value 30 for genes and the fourth column is the values of Linkage LOD score for genes Sc
11. enario 3 Large sample size association test for all chromosomes PLINK files gt Calculate allele frequency gt Generate MERLIN map file gt Check large pedigree gt PedCut gt Split by chrom gt Run association test gt Write results Command line fampipe BED test bed BIM test bim FAM test fam Annotation 1 Anno_file annotation txt Output Please see Association test Output Scenario 4 Imputation only PLINK files gt Calculate allele frequency gt Generate MERLIN map file gt Check large pedigree gt PedCut gt Split by chrom gt Generate intervals from external interval files gt Generate cluster file gt Imputation gt Merge the imputed file Command line fampipe BED test bed BIM test bim FAM test fam Impute 1 Output Please see Imputation output 31 Option Input output option Parameter default Description BED file_name Please see The bed file BIM file name Please see The bim file FAM file name Please see The fam file Freq_file O file name Please see The freq file Cluster file O generate file name Please see The cluster file file name Please see The folder of output 32 Function option Parameter default Description Rutger Map 0 Linkage null npl pairs BD Sharing 0 Impute 0 1
12. erate interval file If you see this message you won t get CHRI range txt as there is no rate greater than Sharing threshold in the IBD Sharing Analysis notable grid chrl 15 Linkage Analysis Linkage analysis function is provided by the Merlin linkage function and FamPipe selects the regions with p values less than Linkage pvalue the default value is 0 05 Command line options Calculate the Whittemore and Halpern NPL all Linkage npl Calculate the Whittemore and Halpern NPL pairs Linkage pairs Related options Specify the threshold of linkage p value default value is 0 05 Linkage_pvalue value For example Linkage_pvalue 0 1 Specify the grid for linkage analysis Default is 1 grid value For example grid 0 5 Memory saving option Turning on this option will significant reduce the memory consumption in Merlin smallSwap PLINK prune threshold variance inflation factor VIF FamPipe uses PLINK to prune SNPs in LD using the VIF SNPs that are not correlated are used for the linkage analysis The default value of VIF is 2 Please see the PLINK manual Linkage disequilibrium based SNP pruning for more information vif value Example vif 1 5 Output Two files CHRX linkage txt and CHRX range txt will be generated for each chromosome CHRX linkage txt saves the Merlin linkage results and CHRX range txt saves the linkage regions each row specifies the
13. ere o a rr cna Fat RR Ne xy anie e gestio PR aa cure a 21 Association test ecce teeex m Ce Re ea eue Dn Le E REL RED EUER en ie ies regar 22 Disease MO Cela gaci mitat M 24 Dis as model type Ain testeemtt emet mic iaeia 24 Disease model type 2 ssssssssssseseseseeee enne enhn nnne nena nnn sns ene 27 Othe options s eb a b ei a bv at de il 29 The Four Commonly Used Scenarios esses esee nnns 30 G HpolE 36 Sugpestlons BUS report cercimsccarnnenniancoaneas utes X Fk PE HRR NUR MEA Pee AME OR QE ARR QYMER RE D ME E ISP RA 37 Introduction FamPipe is a NGS analysis pipeline for family data with complex diseases FamPipe has many commonly used family analysis functions such as identity by descent sharing among affected relatives linkage analysis imputation conditional on family structures disease models and family based association tests Several public resources shown in the following table are integrated in FamPipe Resource Program Function 1000 Genomes Project Data Allele and haplotype frequency estimation Rutger s genetic map Haldane s genetic map positions PLINK2 File conversion PedCut Split large pedigrees for Merlin Merlin IBD sharing linkage and imputation analyses GIGI MORGAN Imputation analysis FBAT Family based association analysis OVPDT Family based association analysis Run the Program Before
14. esence of linkage By default association tests will be performed for all genes provided in the annotation file Alternatively association tests can be performed in linkage regions to save running time This is particularly useful when imputation is performed in linkage regions and association tests are performed based on the imputed data This option should be used with Linkage APL 22 Error Warning Error could not find Anno file If you see this message please check your Annotation chrl txt file whether is in the same folder that you place bed file bim file and fam file Association test Output For example Association chrl txt Gene of SNPs p value TBXI5 354 0 551 There are three columns in the association test result file The first column is the name of genes the second column is the number of snps in genes and the third columns is the association p value 23 Disease model FamPipe offers two types of disease model For the first type of disease model a variant will be predicted as a dominant recessive or none disease mode using the disease model identification DMI algorithm Segregation score and rare heterozygote rule out RHRO mapping status for each variant will also be calculated For the second type of disease model FamPipe checks whether SNPs within a gene follow the rules of compound heterozygosity Command line options The first type of disease model Disease model 1
15. grates several public programs and resources in the analyses The path file specifies the path on the UNIX where each of the program or resource is installed The default path for each program or resource is external The paths can be changed in the path file Command line options path path file name Example path external path txt In the path file several paths can be specified For example assume all executable programs can be found in usr local bin and Rutger s Map can be found in map In the path file plink path usr local bin merlin path usr local bin pedcut path usr local bin fbat path usr local bin ovpdt path usr local bin morgan path usr local bin gigi path usr local bin rutger folder map The External Frequency File Optional Programs like Merlin and GIGI included in FamPipe require a frequency file Therefore for IBD linkage and imputation analyses a frequency file will be generated By default FamPipe estimates allele frequencies from the sample to generate the frequency file If you have a small sample an external file with frequencies estimated based on other samples from the same population can be given to FamPipe Then frequencies in the frequency file for Merlin and GIGI will be estimated jointly based on the sample allele frequencies and external allele frequencies Please see the technical note for the joint estimation of the allele frequencies The format of the external
16. in tutorial Modeling marker marker linkage disequilibrium for more details 19 Merlin Imputation Output merlin infer ped the first five columns are Family ID Individual ID Father ID Mother ID and Sex Others represent each marker for every five columns Most likely genotype Expected Number Alleles P G1 P G2 P G3 For example 11001A A0001G G2100 12002C C2100T T0001 13121A C1010G T1010 merlin infer dat Corresponding to the five columns of each marker in ped file For example M rs77544915 T COUNT 1 1s77544915 C P rs77544915 1 1 C P rs77544915z1 2 C P rs77544915 2 2 M rs2154354 T COUNT 1 1rs2154354 C P rs2154354 1 1 C P rs2154354 1 2 C P rs215435422 2 Merged file X is the chromosome number check infer chrX ped The PLINK format ped file check infer chrX map The PLINK format map file check infer chrX prob the first six columns are the same as the fam file others represent each marker for every three columns probability of genotypes 1 1 2 1 and 2 2 check infer chrX dosage the first six columns are the same as fam file other columns are the Expected Number Alleles for the SNPs 20 GIGI imputation GIGI can use large extended pedigrees for the imputation Therefore pedigrees are not split in GIGI These three input files are required for the GIGI imputation 1 AGWAS file for each chromosome 2 ANGS file for each chromosome 3 Aninterval file for each chro
17. lying the scores for all individuals for the variant RHRO status The RHRO status is determined also based on the algorithm in MendelScan Assuming a dominant model if all affected individuals have Dd at a variant the variant is labeled as SharedHet If at least one affected have Dd and at least one affected have dd the variant is labeled as RareHet The variant 1s labeled as RuleOut for other situations 25 Predicted disease model If Freq score is specified The deminant and recessive mode for a variantis T C Missi TE Missine tolerance thresheld FamPipe will use the DMI algorithm to classify each variant into a disease model autosomal dominant coded as d autosomal recessive coded as r or none coded as n The DMI algorithm needs a threshold file which contains thresholds for different minor allele frequencies The DMI algorithm will classify a variant into a disease model if its segregation score is greater than the threshold with similar minor allele frequency to the variant in the threshold file The thresholds are determined by pedigree structures and therefore should be generated based on the pedigrees you are analyzing Step by step instructions of how to generate a score file can be found here link to the get score file Note if yo
18. markers have been pruned for LD automatically in FamPipe But for imputation analysis a cluster file can be specified By default FamPipe does not use the cluster file However if specified FamPipe can automatically generate the file by estimating the haplotype frequencies in the sample We also compiled a list of the cluster file for the 6 populations with haplotype frequencies estimated from the 1000 Genomes Project Data i e AFR cluster AMR cluster EAS cluster EUR cluster and SAS cluster which can be downloaded from our ftp site The cluster file can also be provided by the user The format of the cluster file is the same as the cluster file from merlin For example CLUSTER snp1165473 snp1165474 snp1165475 HAPLO 0 7662 1 1 1 HAPLO 0 0060 1 1 2 HAPLO 0 0222 1 2 HAPLO 0 0671 2 1 HAPLO 0 1384 2 1 2 Command line options Generate the cluster file based on the sample Cluster_file generate Download the pre compiled cluster file from our ftp site Cluster_file ftp Provide your own file Cluster_file file_name The Interval File Optional As imputation using either Merlin or GIGI for a large region is computationally intensive it is recommended to run the imputation analysis only in linkage regions or in user specified intervals The files containing the user specified intervals should be named as range chrX txt X is the chromosome number Files must include two columns the first column is the s
19. mosome if not running FamPipe with IBD or linkage analysis Please place them in the same folder that you placed the bed file bim file and fam file Please refer to the descriptions of GWAS and NGS SNP List Files in the input file section for the format of the files The interval file can be generated in the same way as described in the previous section Merlin imputation GIGI Imputation Output X is the chromosome number merge chrX ped The PLINK format ped file merge chrX map The PLINK format map file merge chrX prob The first six columns of this file are the same as the Fam file Other columns represent each marker for every three columns which are the probabilities of genotypes1 1 2 1 and 2 2 merge chrX dosage the first six columns are the same as fam file other columns are the Expected Number Alleles for the SNPs 21 Association test Two family based association tests can be performed in FamPipe If imputations were performed FamPipe will use the ped file with the imputed data to run the association tests Command line options Running OVPDT OVPDT is a SKAT type family based association test for rare variants Association 1 Running FBAT FBAT is a burden type family based association test for rare variants Association 2 Related options Anannotation file is required to run the association test Anno file filename Ex Anno_file annotation txt Association in the pr
20. n families with complex diseases Submitted Because different resources are integrated in FamPipe please cite the corresponding papers if you performed analyses based on the following approaches PedCut Liu F et al An approach for cutting large and complex pedigrees for linkage analysis European journal of human genetics EJHG 2008 16 7 854 860 Merlin Abecasis G R et al Merlin rapid analysis of dense genetic maps using sparse gene flow trees Nature genetics 2002 30 1 97 101 GIGI Cheung C Y Thompson E A and Wijsman E M GIGI an approach to effective imputation of dense genotypes on large pedigrees American journal of human genetics 2013 92 4 504 516 FBAT De G et al Rare variant analysis for family based design PloS one 2013 8 1 e48495 OVPDT Chung R H Tsai W Y and Martin E R Family based association test using both common and rare variants and accounting for directions of effects for sequencing data PloS one 2014 9 9 e107800 36 Suggestions Bug report Please contact us if you encounter any difficulties running FamPipe If you would like FamPipe to include your software or would like to suggest new functions in FamPipe you are also welcome to contact us The email should be sent to rchung nhri org tw 37
21. ng Merlin imputation Imputation will only be performed within the intervals defined in the interval file There are two ways to provide the interval file 1 Generated by FamPipe after executing Linkage Analysis or IBD Sharing Analysis The interval files will be automatically used for the GIGI imputation Note If you see an error message Error could not find interval file that means there were no intervals that met the threshold of Linkage pvalue or Sharing threshold 2 Provided by the user See The interval file in the input file section 18 Related options Theusercan provide a cluster file to account for LD for the imputation Cluster file file name Ex Cluster file merlin clusters Ifthe sample size is large haplotype frequencies can be estimated automatically based on the samples in FamPipe FamPipe uses the Merlin options rsq 0 1 cfreq to generate the cluster file Cluster file generate The haplotype frequencies estimated based on the 1000 Genomes Project can be downloaded from our ftp site Cluster file ftp Note if running Merlin Imputation without any cluster file you will see WARNING Could not open file CHRI clusters with clustering information on console Also please note that if the cluster file is not generated based on the samples markers in the haplotype that is observed in the sample but not in the cluster file are dropped by Merlin Please refer to the Merl
22. s separately and also provide additional information for inbred pedigrees Notable grid file The file saves the positions with the proportions of IBD pairs in all possible pairs of affected individuals Example notable grid chrl position 148 108 rate 0 28 count 35 num of pair 125 position 149 108 rate 0 28 count 35 num of pair 125 The position corresponds to the genetic position in the bim file Assume the value of grid is 1 Therefore the position from row to row increases by 1 Rate is the proportion of IBD pairs in all pairs of affected individuals The number of pairs is the number for all possible pairs of affected individuals excluding parent offspring pairs Range file The file contains the chromosome regions where the proportion of IBD pairs Sharing threshold Each row specifies one region with the start and end physical positions For example CHRI range txt 118992458 119203063 If CHRI range txt file is not generated it means that no positions met the Sharing threshold Annotation file If an annotation file is provided using the Anno file option the IBD statistics will be added as extra columns in the annotation file 14 Error Warning Warning could not find the result in IBD Sharing Analysis If you see this message the notable grid files were not generated Warning There is no IBD rate larger than Sharing threshold threshold in notable grid chrl that will not gen
23. start and end physical positions with p values less than Linkage pvalue 16 Error Warning Warning could not find the result in linkage Analysis If you see this message it means Merlin did not generate results Warning There is no pvalue smaller than Linkage pvalue threshold in CHRI linkage txt Interval file will not be generated If you see this message you won t get CHRI range txt as there is no pvalue less than Linkage pvalue threshold in Linkage output file CHR1_linkage txt For more information about the linkage analysis in Merlin please see MERLIN Tutorial Linkage Analysis http www sph umich edu csg abecasis merlin tour linkage html 17 Imputation Analysis FamPipe provides imputation analysis based on two popular family based imputation tools Merlin and GIGI Command line options Impute 1 Merlin Imputation Impute 2 GIGI Imputation Merlin Imputation Merlin Imputation is performed using the infer option in Merlin PedCut is automatically executed by FamPipe to split large pedigrees into sub pedigrees with the bit size specified by the user After imputation the sub pedigrees are merged based on the original pedigree structures If an individual is in several sub pedigrees the individual may be imputed multiple times During the merge concordance calls for the individual are reported and others are set as missing An interval file for each chromosome is required for runni
24. tart of the physical position and the second column is the end of the physical position for each interval For example In range chrl txt 118754941 119491686 119486998 119491486 In this example two intervals are defined on chromosome 1 and imputations will only be performed in these intervals Note please name the interval files for each chromosome as range chr1 txt range chr2 txt and place them in the same folder as the one you place the bed bim and fam files Command line options interval The GWAS and NGS SNP List Files Required for running GIGI GIGI requires a sparse set e g the GWAS SNPs and a dense set e g the NGS SNPs of markers for imputation Therefore SNP sets for each chromosome should be provided before running GIGI These files should be saved in the same folder where you launched FamPipe The SNP set file has one column where each row is the name for each SNP in the set The GWAS SNP files should be named as gwas snpsX txt where X is the chromosome number The NGS SNP files should be named as ngs snpsX txt where X is the chromosome number For example the GWAS and NGS SNP files for chromosomes 1 and 2 are gwas snpsl txt gwas snps2 txt ngs snpsl txt ngs snps2 txt The content of the file is like gwas snpsl txt snp1149977 snp1149980 snp1149981 snp1149997 snp1149998 ngs snpsl txt snp1149972 snp1149977 snp1149978 snp1149980 snp1149981 The Annotation File Requir
25. the genetic positions By default FamPipe reads the genetic positions from the third column in the bim file The positions can also be updated based on the Rutger s Map The values in the third column genetic positions in the bim file will be updated using this function More details of how FamPipe updates the positions can be found in the technical note The latest version of Rutger s Map can be downloaded from http compgen rutgers edu download_maps shtml The map file for each chromosome should be named as Rutger Map chrX txt where X is the chromosome number Also please make sure the path for rutger folder in the external program path file points to the folder where the map files are placed Note 1 If you use this function there must be more than one SNP in the bim file that have identical names in Rutger s Map 2 If you use your own genetic positions in the bim file please follow the rules for the bim file The genetic positions in the third column cannot be duplicated Every value of the genetic position must be less than 500 Command line options Rutger Map Output An updated map new XXX bim will be saved in the output file folder 11 PedCut FamPipe automatically splits large pedigrees using PedCut in order to run Merlin For more information about the PedCut algorithm please refer to the PedCut user manual which can be downloaded at http mga bionet nsc ru soft pedcut pedcut 1 19 tar gz Ou
26. tput In the data output folder you will find the file family pedcut info which contains the information of the PedCut results For example the file may look like 1 345 2 6789 There are two pedigrees in the fam file The first pedigree is split into 3 sub pedigrees with family IDs 3 4 and 5 and the second pedigree is split into 4 sub pedigrees with family IDs 6 7 8 and 9 Families are recoded with the new IDs for the Merlin analyses Related options Specify the bit size for PedCut and Merlin The bit value is calculated as two times the number of non founders minus the number of founders The default value is 20 which worked in our server with 96 GB of memory You may reduce the value to save memory or turn on the smallSwap option bit value Example bit 15 12 IBD Sharing Analysis IBD Sharing Analysis is based on the Merlin IBD Estimation using the extended option in Merlin FamPipe identifies the regions that have higher IBD sharing among affected individuals A pair of affected individuals who can be traced to a common ancestor and who have P IBD 0 for a region greater than IBD threshold is defined as an IBD pair for the region Parent offspring pairs are not considered as they always share 1 allele IBD The proportion of IBD pairs in all pairs of affected individuals excluding parent offspring pairs is calculated for each variant For more details about how FamPipe defines IBD sharing regions please
27. u only have a few pedigrees please consider using external allele 26 frequency file e g by specifying the Freq file so that D and d can be properly determined Output The output of the disease model shows the predicted disease model status segregation scores and RHRO status of every marker For example valid marker chr1 marker name Predicted model Dominant Segregation Score Recessive Segregation Score RHRO snplr 4 97323e 33 1 RuleOut snp2n 1 26218e 29 1e 96 RuleOut snp6n 60 25e 45 3 55271e 60 RaredHet snp7n 1 03398e 27 1 2207e 87 RaredHet snp8n 1 26218e 29 1e 96 RuleOut Disease model type 2 The five rules proposed by Kamphans et al PLOS ONE 2013 with some relaxation based on different thresholds were used to determine whether SNPs in a gene follow compound heterozygosity 1 A variant has to be in a heterozygous state Dd in at least compound rule1 100 46 of affected individuals The proportion of affected people who match this condition must be greater than compound rulel 2 A variant must not occur in a homozygous state in at least compound rule2 100 90 of the unaffected individuals The proportion of 27 unaffected people who match this condition must be greater than compound rule2 3 A variant that is heterozygous in an affected child is heterozygous in exactly one of the parents At least compound rule3 100 96 of affected children must follow this
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