Supporting Information - yin.hms.harvard.edu

1 Supporting InformationBeliveau et al. of features available in OligoArray and et of blockParse. (A) Schematic diagram illustrating the nature and order of checks used to screen candidate probe sequences. (B) De-scription of command-line options and the default values/settings for et of outputClean. (A) Schematic diagram illustrating the task order of the script. (B) Description of command-line options and the defaultvalues/settings for et Information for each temperature-specific LDA model. (A F) For each temperature, the precision, recall, support-weightedF1score, andsupport are et (interval size kb)log10(time s)01234567 OligoArray: y= + , R2 = : y= + , R2 = 40,000,001 - 43,000,000 OligoArrayOligoMiner8,733 ,11358,9440100200300400500 Probes/kb OligoArray0100200300400500600700 Probes/kb OligoMinerR2 = 11,000,001 - 14,000,000 OligoArrayOligoMiner15,078161,51840,0670 100200300400500 Probes/kb OligoArray0100200300400500600 Probes/kb OligoMinerR2 = 149,000,001 - 152,000,000 OligoArrayOligoMiner29,036263,39262,4890 100 200 300 400 500 600 Probes/kb OligoArray0100200300400500600700 Probes/kb OligoMinerR2 = and coverage comparison between OligoArray and OligoMiner.

2 (A) Mean times SD for probe discovery in eight 10-kb, 100-kb, 1-Mb, or 10-Mbintervals using OligoArray and OligoMiner. The fold increase in speed provided by OligoMiner for each interval is also shown. (B) Log-log plot of the data regression trend lines, equations, andR2values are also shown; shading represents the 95% CI of the fit. (C E) Venn diagrams (Left) and plots showinglinear regressions (Right) of three 3-Mb intervals mined exhaustively using OligoArray and OligoMiner. Plots show the number of probes discovered in eachnonoverlapping 1-kb window starting with the first coordinate of each interval. Linear regression trend lines andR2values are also shown; shading represents the95% CI of the et plots depicting the duplexing probabilities of all kmers 8 nt in length with the reverse complements of their 40 46mer parental sequences at sixdifferent simulation temperatures in 390 mM Na+and 50% et S1.

3 Description of Oligopaint probe sets usedProbe setTargetSize, kb ComplexityDensity, probesper kilobaseMining parametersSynthesis methodXq28 hg38 chrX:149,681,511 151,005,6771,3245, 45mers, 47 52 C Tm; UM, no kmerFilterGel extractionXq28 hg38 chrX:151,015,314 153,048,8062,0336, 45mers, 47 52 C Tm; UM, no kmerFilterGel extractionXq28 hg38 chrX:153,051,165 153,868,1518174, 45mers, 47 52 C Tm; UM, no kmerFilterGel hg19 chr19:9,921,047 11,719,2071,79810, 41mers, 42 47 C Tm; 47 C LDM,kmerFilter -m 18 -k hg19 chr19:11,725,035 12,760,0261,0353, 41mers, 42 47 C Tm; 47 C LDM,kmerFilter -m 18 -k kbhg19 chr19:13,689,983 13,709, 41mers, 42 47 C Tm; 47 C LDM,kmerFilter -m 18 -k 5T7 Xist RNAhg38 chrX:73,841,382 73,852, 41mers, 42 47 C Tm; 42 C LDM,kmerFilter -m 18 -k 5 Commercial columnsynthesisTable S2.

4 Description of oligo sequences used for probe set generation and visualizationSequence 5 -3 PurposeProbe sets used withATTO488-CCAGTGCTCGTGTGAGAAGTCPCR primerXq28 CTGCAGAGAAGAGGCAGGTTCPCR primerXq28 ATTO565-CGCTCGGTCTCCGTTCGTCTCPCR primerXq28 GGGCTAGGTACAGGGTTCAGCPCR primerXq28 Alexa647-CAGGTCGAGCCCTGTAGTACGAPCR primerXq28 ATTO488-TTGATCTACATATTCAGGTCGAGCCCTGTAGT ACGPCR primerXq28 CTAGGAGACAGCCTCGGACACPCR primerXq28 TATGTAGATC-CY3 BDNA-PAINTXq28 TTATACATCTAGDNA-PAINTXist RNACTAGATGTAT-CY3 BDNA-PAINTXist RNACCATGGCGAGAAGGTCTGCGPCR primer , , chr19 20 kbTAATACGACTCACTATAGGGGCGTGTGCGAGTGGTTGG ACPCR primer+T7 , , chr19 20 kbGTGTACCGCGATCCGAAGCGGGTCTTACAGCGGCGCAA TGTTCACACGCTCTCCGTCTTGGCCGTGGTCGATCAS econdary Alexa647-TGATCGACCACGGCCAAGACGGAGAGCGTGT GAGATGTTT-Alexa647 Tertiary GGTGTGGGCTAGCGCCAATCGGTCTTACAGCGGCGCAATG TTTAGCGCAGGAGGTCCACGACGTGCAAGGGTGTS econdary CY3B-ACACCCTTGCACGTCGTGGACCTCCTGCGCTA-CY 3 BTertiary Alexa647-ACACCCTTGCACGTCGTGGACCTCCTGCGCT ATTTTTTTT-Alexa647 Tertiary CTCCGGCGGACTCATCCCAGGGTCTTACAGCGGCGCAATG TTCACCGACGTCGCATAGAACGGAAGAGCGTGTGS econdary 20 kbAlexa647-CACACGCTCTTCCGTTCTATGCGACGTCG GTGTTTTTTTT-Alexa647 Tertiary 20 kbAlexa405-CATTGCGCCGCTGTAAGACCT ertiary &20kbDataset S1.

5 OligoMiner readme fileDataset S1 Dataset S2. Temperature-dependent differences in off-target discrimination potentialDataset S2 Beliveau et