Peak scanner 2

Files from the Genetic Analyzer were opened in Peak Scanner 2 software (
http://www.appliedbiosystems.com) using Analysis Method=Sizing Standard-PP, and Size standard=GS500. Only the green (hex-primer-labeled) peaks were used to determine level of bacterial abundance. The size and heights of the green peaks were calculated using the ROX GS500 standard. The individual output files were assembled into a single microbial profile file using the RiboSort package
^{34 (link)} using the script, RiboSort(data,dataformat="standard",dye="G", output="proportions",zerorows=FALSE,repeats=1, mergerepeats="presentinall"). This program automatically bins the peaks to generate abundances; we used the default threshold of 50 fluorescence units as recommended by the program authors.

Genomic stability was confirmed by amplification of 6 different STR markers following standard protocols and analysis on a Sequence Analyser 3130XL (Applied Biosystems, Foster City, CA, USA). Fragment sizes were analyzed with PeakScanner 2 software (Applied Biosystems, Foster City, CA, USA).

DNA was extracted from ear biopsies using a Qiagen DNeasy® extraction kit and subsequently genotyped at six MHC Class I linked microsatellite loci described by Cheng & Belov^{70 (link)}; Sh-MHCI101, Sh-MHCI102, Sh-MHCI105, Sh-MHCI106, Sh-MHCI107, and Sh-MHCI108. For further details on size range and primer sequences see Cheng & Belov^{70 (link)}. Polymerase chain reactions (PCR) were carried out using the protocols of Cheng & Belov^{70 (link)} and the PCR products were quality tested on a 2% agarose gel. The samples were analyzed at the Ramaciotti Centre (University of New South Wales, Australia) and microsatellite profiles of the individual devils were subsequently scored using Peak Scanner 2.0 (Applied Biosystems 2012).

Genomic DNA (gDNA) for both species was isolated using the HotShot protocol (Truett et al., 2000) specifically designed for small amount of tissues (method described in Appendix S1). Samples were screened at 10 microsatellite loci per species (Table S3). Details of PCR and profiling are provided in Appendix S1.
Fragment analysis was carried on an ABI 3730 DNA Analyzer (Applied Biosystems), and genotype peaks were scored using Peakscanner 2.0 (Applied Biosystems). Allele calling was performed with FlexiBin v2 (Amos et al., 2006). Each genotypic dataset was checked for quality in terms of (a) missing data, (b) repeatability, and (c) scoring errors (stuttering bands, allele dropouts, and null alleles). Repeatability was checked by re‐calling alleles for a subset of randomly chosen samples (~10% per dataset) and comparing them with previous scoring. The possibility for scoring errors was tested via Microchecker 2.2.3 (van Oosterhout, Hutchinson, Wills, & Shipley, 2004) and data corrected for null alleles using the Brookfield algorithm (Brookfield, 1996).

XCI patterns were examined using a methylation-sensitive PCR-based assay targeting the human X-linked androgen receptor gene (HUMARA) locus as described previously^{33 (link),34 (link)}. Genomic DNA digested with methylation-sensitive restriction enzyme HpaII (digested) and undigested DNA (undigested) were used as templates to amplify HpaII sites of the first exon of the HUMARA gene that contained the highly polymorphic repeat sequence by PCR using primers flanking the locus (Supplementary Table 1). The inactivation rate was calculated using PeakScanner2 (Thermo Fisher Scientific).

Bidirectional triplet-primed PCR (TP-PCR) followed by capillary electrophoresis on an ABI 3730 DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) was used to assess patients for the presence of interruptions in the 3′ and 5′ ends of the CTG repeat. The results were visualized using Peak Scanner 2 (Thermo Fisher Scientific). Repeat interruptions are usually stretches of CCG or CGG repeats towards the 3′ end of the CTG repeat [24 (link),25 (link)], although other interruptions such as CTC [24 (link),26 (link)], CAG [27 (link)], and GCC [28 (link)] have also been reported, and interruptions have been observed at both the 5′ and 3′ ends of the repeat [27 (link)]. Repeat interruptions can be identified as gaps in the continuous and decreasing pattern of peaks seen when the output of TP-PCR is analyzed (Figure S1). The primer sequences, modifications to the manufacturer’s instructions, and PCR conditions can be found in Table S1.

Samples positive for two or more quality verification nuclear markers were used for microsatellite analysis. Twelve validated highly polymorphic microsatellite markers (MS1, MS2, MS5, MS6, MS7, MS8, MS9, MS10, MS12, MS15, MS20 and MS3.27) were amplified using hemi-nested PCR^{27 (link)}. Each 10-μL reaction consisted of 5 μL of the PrimeSTAR Max Premix (Takara-Bio, Kusatsu, Japan) and 1.25 μM of each primer (Supplementary Data 1). The primary PCR used 3 μL of DNA as a template, while the nested PCR used 1 μL of the primary PCR product. The cycling conditions for the primary PCR were as follows: 95 °C for 1 min; 40 cycles at 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 5 min. The cycling conditions for the nested PCR were as follows: 95 °C for 1 min, 35 cycles at 95 °C for 15 s, 61 °C for 30 s, 72 °C for 45 s and a final extension at 72 °C for 5 min. The nested PCR products were resolved on an Applied Biosystems 3730xl DNA Analyzer using the GeneScan 600 LIZ size standard (Thermo Fisher Scientific). Allele sizes were determined using Peak Scanner 2 (Thermo Fisher Scientific). Alleles with a minimum of one-third of the relative fluorescence units of the primary allele were recorded as secondary alleles.