Supplementary Information: Linked-read sequencing for detecting short tandem repeat expansions


Four and six reference lymphoblastoid cell lines with GAA-and CGG-repeat expansions in the frataxin (FXN) and fragile X mental retardation 1 (FMR1) genes, respectively, were obtained from the Coriell Cell Repository (Camden, New Jersey, United States; https://www.coriell.org/). 

mutation/FM), and repeat lengths reported by Coriell are shown in Supplemental Table 1. High molecular weight genomic DNA derived from the cell lines was used for library preparation with the Chromium Genome Reagent Kit (v2 Chemistry). Paired-end whole-genome sequence (WGS) reads of 150 base pairs were generated on an Illumina HiSeq X instrument and processed using the Long Ranger™ pipeline (v2.2.2) from 10x Genomics. The generated reads were mapped to the hg19 human reference genome build. 
Table S1. FMR1 and FXN genotypes and allelic repeats lengths of Coriell samples used for 10x linked-read whole-genome sequencing. The two normal samples, GM06889 and GM06895, were not analyzed using the barcode-based methods presented in the manuscript. On-line information to the cell lines is accessible through the hyperlinks under "Coriell ID". Benchmarking based on collecting JI profiles from 50,000 locations at distances from 200 to 10,000 at a step-size of 100 bp (step 1) and generating estimates for 13 loci (step 2) on NA12878 TELL-Seq data (1.11B reads). 

Coriell ID

Step 1 was run with multi-processing using 42 parallel processes. 10x and stLFR simulation 10x and stLFR WGS with repeat expansions were simulated using LRSim 1 (v1.0) and stLFR read simulator 2 , respectively. The ATXN10, ATXN8OS, GIPC1, GLS, NOP56, RFC1, C9ORF72, SAMD12, and FMR2 repeat loci in the hg38 human reference genome were modified to contain expanded stretches of repeats (shown in Table S2a) using reform 3 . The modified and unmodified reference FASTA files were processed using LRSim's simulateLinkedReads.pl script in -r (reference) mode: perl simulateLinkedReads.pl -r $modFASTA -p $output_prefix -7 0 -0 0 -z 16 perl simulateLinkedReads.pl -r $unmodFASTA -p $output_prefix -7 0 -0 0 -z 16 "clean" FASTA files from the modified and unmodified reference FASTA were used as inputs with the simulateLinkedReads.pl and stLFRSim to generate 10x and stLFR reads as follows: 
perl simulateLinkedReads.pl -g hg38.hap.0.clean.fasta,hg38.hap. The generated paired-end 150 base pair 10x reads from the modified and unmodified sequences were concatenated and processed using Long Ranger: longranger wgs --id=sim${i} --fastqs=$fq --reference=$PATH/reference_genomes/H_sapiens/10X-Genomics/refdata-GRCh38-2.1.0 --localcores=30 --localmem=120 --vcmode=freebayes --sex=female 
The generated paired-end 100 base pair stLFR reads from the modified and unmodified sequences were concatenated and mapped to the hg38 reference genome using BWA-MEM 4 , duplicates in the BAM file marked with Picard 5 , and indexed using SAMtools 6 . 

Fig. S1 .

 S1 Fig. S1. A comparsion of genotyping performance on simulated STR expansions in BLRS data between ExpansionHunter (EH), barcode-based in-repeat reads (IRR), and Jaccard index (JI) methods. Black vertical bars indicate repeat counts of loci in reference genome. Orange vertical bars indicate repeat counts of simulated alleles. EH_noOTS: EH analysis without off-target sites (OTS) and EH_OTS: EH analysis with OTS. 


Fig. S2 .

 S2 Fig. S2. A comparsion of genotyping performance on STR expansions, relative to the human genome and verified by de novo assembly, in NA12878 BLRS data between ExpansionHunter (EH), barcode-based in-repeat reads (IRR), and Jaccard index (JI) methods. Black vertical bars indicate repeat counts of loci in reference genome. Orange vertical bars indicate repeat counts of alleles determined from sequence assembly. "NA" indicates results not available because of failed software run or software's failure to genotype specific locus. EH_noOTS: EH analysis without off-target sites (OTS). 


Table S2a .

 S2a Information of modified loci in 10x and stLFR simulations used for method comparison. 


	Gene	Chromosome	Motif	Size (bp)	Copy number Zygosity	Sex
	GIPC1	19	CCG	500	167	het	male
	ATXN8OS	13	CTG	750	250	het	male
	FMR2	X	CCG	1000	334	hemi	male
	C9orf72	9	GGCCCC	1500	250	het	male
	SAMD12	8	TGAAA	2000	400	het	male
	GLS	2	GCA	2500	834	homo	male
	GLS	2	GCA	3000	1000	homo	male
	RFC1	4	AAGGG	3500	700	homo	male
	RFC1	4	AAGGG	4500	900	homo	male
	NOP56	20	GGCCTG	5000	834	het	male
		Actual genotype	Gene	Repeat motif	Repeat size (copy number)
	GM15847 GM15848 GM15849 GM15850 GM06889 GM06891 GM06894 GM06895 GM06896 GM06897 used for method comparison. NL/FM NL/FM NL/FM FM/FM NL/NL PM NL/PM NL NL/PM FM Locus chr11:133354203-133354353 chr11:92705289-92706006 chr13:112972218-112972863 chr13:21725858-21726151 chr18:22231934-22232859 chr18:77252855-77253057 chr4:5706294-5706480 CGG Table S2b. Information of expanded (relative to reference genome) loci in NA12878 assembly FXN GAA NL/760 FXN GAA NL/830 FXN GAA NL/920 FXN GAA 650/1030 FMR1 CGG 23/30 FMR1 CGG 118 FMR1 CGG 30/78 FMR1 CGG 23 FMR1 CGG 23/95-120-140 FMR1 Motif Reference Size (bp) Assembly sizes (allele1/allele2 bp) TGG 151 1715/2790 GTG 718 2437/2411 TGGA 646 1612/1612 CACCAC 294 2990/3069 TCCA 926 2207/2331 CAC 203 1496/506 ATAG 187 2456/3136 chr4:7724106-7725052 GTG 947 1269/1627
	chr4:8294583-8295012	TCCA	430	1640/1640
	chr6:3221957-3222326	CAC	370	1631/1600
	chr6:834343-834462	TCCT	120	1843/1876
	chr7:141336723-141337391	CCA	669	1962/1515
	chr7:98810989-98811637	GTG	649	3389/3385
	het: heterozygous; homo: homozygous; hemi: hemizygous		

Table S3 .

 S3 Computing benchmarks of both IRR and JI methods. Computing was performed on an Intel Xeon Gold 6254 3.10 GHz 144-core machine running Centos 7.6. 


	Method	Step	Time	Memory (Peak)
	IRR	1. Extract reads based on barcodes mapped to target region (extract_irr.py)	36m:04s	550.79 MB
		2. Identify IRR reads from extracted read set (id_irr.py)	4m:53s	1.73 GB
		3. Determine repeat size based on IRR counts, read length, and coverage (get_irr_size.py)	14.35s	82.71 MB
		Benchmarking based on genotyping 13 loci on NA12878 stLRF data (3.34B reads)
	JI	1. Generate database of JI profiles for varying		
		genomic intervals at random locations	14m:11s	32.25 GB
		(model_span.py)		
		2. Match JI profile of target region against		
		database to estimate target's genomic distance	18.645s	964.44 MB
		(estimate_span.py)		

The FXN and FMR1 reference samples, their genotypes (normal/NL, premutation/PM, and full- 


EH analysis with OTS

The EH variant catalog file used for ATXN10 analysis (hg38) is: { "OffTargetRegions": [ "chrX:9791969-9792159" ], "RepeatId": "ATXN10-withOTS", "RepeatUnit": "ATTCT", "TargetRegion": "chr22:45795355-45795424" } 
The EH variant catalog file used for FXN analysis (hg19) is: { "OffTargetRegions": [ "chr1:102123410-102123560", "chr13:102813926-102814076", "chr2:221410907-221411057", "chr5:126583029-126583179", "chrX:51364391-51364541" ], "RepeatId": "FXN-withOTS", "RepeatUnit": "GAA", "TargetRegion": "chr9:71652203-71652220" } 
The EH variant catalog file used for FMR2 analysis (hg38) is: { "OffTargetRegions": [ "chr17:32142451-32142503", "chr19:10871563-10871651", "chr2:86914348-86914529", "chr7:100694312-100694385", "chrX:19990922-19990974", "chrX:67546512-67546566", "chrX:150983398-150983456" ], "RepeatId": "FMR2", "RepeatUnit": "CCG", "TargetRegion": "chrX:148500638-148500683" } 
The EH variant catalog file used for GIPC1 analysis (hg38) is: { "OffTargetRegions": [ "chr17:32142451-32142503", "chr19:10871563-10871651", "chr2:86914348-86914529", "chr7:100694312-100694385", "chrX:19990922-19990974", "chrX:67546512-67546566", "chrX:150983398-150983456" ], "RepeatId": "GIPC1", "RepeatUnit": "CCG", "TargetRegion": "chr19:14496041-14496074" } 
The EH variant catalog file used for GLS analysis (hg38) is: { "OffTargetRegions": [ "chr16:73546662-73546734", "chr17:10995712-10995773", "chr17:51831668-51831732", "chr18:55586153-55586230", "chr19:45770206-45770266", "chr3:149766818-149766881", "chr7:7673311-7673371", "chrX:67545316-67545385" ], "RepeatId": "GLS", "RepeatUnit": "GCA", "TargetRegion": "chr2:190880872-190880920" } 
The EH variant catalog file used for NOP56 analysis (hg38) is: { "OffTargetRegions": [ "chr5:146459014-146459174" ], "RepeatId": "NOP56", "RepeatUnit": "GGCCTG", "TargetRegion": "chr20:2652733-2652757" } 
The EH variant catalog file used for RFC1 analysis (hg38) is: { "OffTargetRegions": [ "chr10:25874364-25874549", "chr10:100315011-100315116", "chr11:63257658-63257763", "chr21:39583818-39584123", "chr5:79180390-79180505", "chr5:79180514-79180629", "chrX:48280812-48280912" ], "RepeatId": "RFC1", "RepeatUnit": "AAGGG", "TargetRegion": "chr4:39348424-39348479" } 
The EH variant catalog file used for SAMD12 analysis (hg38) is: { "OffTargetRegions": [ "chr21:34718631-34718684", "chr3:151368620-151368760", "chr4:114319040-114319089", "chr4:122718712-122718762", "chr6:157534533-157534600", "chr8:139313899-139313962", "chr9:78008566-78008623" ], "RepeatId": "SAMD12", "RepeatUnit": "TGAAA", "TargetRegion": "chr8:118366913-118366914" } 
The EH variant catalog file used for C9ORF72 analysis (hg38) is: { "OffTargetRegions": [ "chrX:154390486-154390564" ], "RepeatId": "C9ORF72", "RepeatUnit": "GGCCCC", "TargetRegion": "chr9:27573528-27573546" } Generation of OTS: To identify the off-target sites, we simulated 100 or 150 bp reads entirely composed of the "motif of interest" and mapped these reads to the reference genome (hg19 or hg38) using bwa mem. The mapping coordinates of the reads were extracted from the sorted BAM files using bedtools and were provided as off-target sites to EH. 

Illumina Coriell genomes

The Illumina Coriell genomes 7 analysed in this study can be accessed in the European Genomephenome Archive repository: https://www.ebi.ac.uk/ega/datasets/EGAD00001003562.
Readman Chiu, Indhu-Shree Rajan-Babu, Inanc Birol, Jan M Friedman
Supplementary Information: Linked-read sequencing for detecting short tandem repeat expansions