DOI: 10.5061/dryad.18931zd5z
title: Supplemental data from: Next-generation sequencing base calls for mosaic mutations

description: Context: Some children with diazoxide-unresponsive congenital
 hyperinsulinism (HI) lack any detectable disease-causing mutation in
 peripheral blood DNA.  Objective: To examine whether somatic
 post-zygotic mutations of known HI genes are responsible for disease in
 children with diazoxide-unresponsive HI requiring surgery with histology
 not classified as focal or LINE, and without detectable mutations by
 standard genetic testing of peripheral blood DNA.  Methods:
 Next-generation sequencing (NGS) was performed on specimens of pancreas
 from 10 children with diazoxide-unresponsive HI. Results: Four unique GCK
 mutations were identified at low levels of mosaicism ranging from
 4.4-10.1% in pancreatic DNA from five of these 10 children.  The
 GCK mutations were not detectable in peripheral blood DNA by NGS in three
 cases from which peripheral blood DNA was available for testing. 
 All four GCK mutations have been previously published as activating HI
 mutations.      Conclusion: These
 results indicate that post-zygotic somatic mutations of known HI genes are
 responsible for some cases of diazoxide-unresponsive hyperinsulinism.
description: Next Generation Sequencing:  Pancreatic islets were isolated from
 surgical specimens as previously described (20,21). DNA and RNA were
 isolated from peripheral blood and pancreatic tissue using standard kits
 following manufacturer’s instructions.  Exonic and flanking intronic
 regions of <em>ABCC8, KCNJ11, </em>and<em>
 GCK</em>, were PCR amplified and combined in equimolar
 concentrations prior to NGS (Ion Torrent, Life Technologies) as previously
 described (14).  In order to detect low-level mosaic mutations, NGS was
 performed with a high depth of coverage.  The average depth of coverage
 for the cases included here was 17,612x compared to ~100x that is
 typically offered on standard clinical testing (22). Signal processing and
 base calling were performed using the Torrent Suite Software, followed by
 alignment with the Torrent Mapping Alignment Program (TMAP). Variant
 calling and coverage analysis were performed on aligned reads using the
 Torrent Variant Caller and Coverage Analysis plugins (Life Technologies,
 Carlsbad, CA). Variant calling included a low stringency filter to
 identify mosaic variants as low as 2% using the “somatic low stringency”
 configuration in the Torrent Variant Caller plugin.
 <em>GCK</em> sequence information is based on GenBank
 reference sequence NM_000162.3.  Variants were searched against the gnomAD
 Browser (v2.1) (23).  Variants were confirmed by direct sequencing as
 previously described (1).
description: # Supplemental Table. Next-Generation Sequencing base calls for mosaic
 mutations.
 [https://doi.org/10.5061/dryad.18931zd5z](https://doi.org/10.5061/dryad.18931zd5z) ## Description of the data and file structure Next Generation Sequencing:  Pancreatic islets were isolated from surgical specimens of children with diazoxide-unresponsive hyperinsulinism as previously described (20,21). DNA and RNA were isolated from peripheral blood and pancreatic tissue using standard kits following manufacturer’s instructions.  Exonic and flanking intronic regions of *ABCC8, KCNJ11, and* GCK, were PCR amplified and combined in equimolar concentrations prior to NGS (Ion Torrent, Life Technologies) as previously described (14).   In order to detect low-level mosaic mutations, NGS was performed with a high depth of coverage.  The average depth of coverage for the cases included here was 17,612x compared to ~100x that is typically offered on standard clinical testing (22). Signal processing and base calling were performed using the Torrent Suite Software, followed by alignment with the Torrent Mapping Alignment Program (TMAP). Variant calling and coverage analysis were performed on aligned reads using the Torrent Variant Caller and Coverage Analysis plugins (Life Technologies, Carlsbad, CA). Variant calling included a low stringency filter to identify mosaic variants as low as 2% using the “somatic low stringency” configuration in the Torrent Variant Caller plugin. *GCK* sequence information is based on GenBank reference sequence NM_000162.3.  Variants were searched against the gnomAD Browser (v2.1) (23).  Variants were confirmed by direct sequencing as previously described (1). ### Files and variables #### File: Supplemental\_Table\_NGS\_base\_calls\_for\_mosaic\_mutations\_010323.xlsx This data set includes the base calls for the mosaic GCK mutations in all reads, as well as the base calls for the five nucleotides upstream and downstream from the mosaic mutation in three cases.  In each case, base calls are included for all tissues tested, including peripheral blood DNA, cell line DNA, and/or DNA from isolated islets and/or whole pancreas.  The nucleotide change associated with the mosaic GCK mutation in each case is outlined in a bolded boarder and bolded font.  Red highlighting represents the mosaic GCK mutation call that is above the background mis-call rate.  Variables: *       Case: Case ID *       Gene: Nucleotide and Amino Acid change of the identified GCK mosaic mosaic mutation in each case. *       Sample Type: source of DNA *       Nucleotides: all possible nucleotides at each position *       -5bp: Nucleotide calls 5 bases upstream from the mosaic mutation.  The nucleotide calls are listed both as total calls in all reads at that position (column E) as well as in percentages (column F).    *       -4bp: Nucleotide calls 4 bases upstream from the mosaic mutation.  The nucleotide calls are listed both as total calls in all reads at that position (column G) as well as in percentages (column H).     *       -3bp: Nucleotide calls 3 bases upstream from the mosaic mutation.  The nucleotide calls are listed both as total calls in all reads at that position (column I) as well as in percentages (column J).       *       -2bp: Nucleotide calls 2 bases upstream from the mosaic mutation.  The nucleotide calls are listed both as total calls in all reads at that position (column K) as well as in percentages (column L).     *       -1bp: Nucleotide calls 1 base upstream from the mosaic mutation.  The nucleotide calls are listed both as total calls in all reads at that position (column M) as well as in percentages (column N).     *       Mosaic Mutation: Nucleotide calls for the identified mosaic GCK mutation in each case.  The nucleotide calls are listed both as total calls in all reads at that position (column O) as well as in percentages (column P). The nucleotide change (total calls and percentage of calls) associated with the mosaic GCK mutation in each case is outlined with a bolded boarder and bolded font.  Red highlighting represents the mosaic GCK mutation call that is above the background mis-call rate.       *       +1bp: Nucleotide calls 1 base downstream from the mosaic mutation.  The nucleotide calls are listed both as total calls in all reads at that position (column Q) as well as in percentages (column R). *       +2bp: Nucleotide calls 2 bases downstream from the mosaic mutation.  The nucleotide calls are listed both as total calls in all reads at that position (column S) as well as in percentages (column T).    *       +3bp: Nucleotide calls 3 bases downstream from the mosaic mutation.  The nucleotide calls are listed both as total calls in all reads at that position (column U) as well as in percentages (column V).      *       +4bp: Nucleotide calls 4 bases downstream from the mosaic mutation.  The nucleotide calls are listed both as total calls in all reads at that position (column W) as well as in percentages (column X).  *     +5bp: Nucleotide calls 5 bases downstream from the mosaic mutation.  The nucleotide calls are listed both as total calls in all reads at that position (column Y) as well as in percentages (column Z).    ## Code/software This data set is formatted as an .xlsx file and requires Microsoft Excel or a similar program to view. ## Access information Other publicly accessible locations of the data: * n/a Data was derived from the following sources: * n/a