MRD in a Mouse Model of RUNX1-mutated CN-AML
Our aim was to determine if any new sequence variations occurred in CG-SH post-engraftment in genes known to be involved in myeloid malignancies.  1e6 CG-SH cells from culture were engrafted into three different non-irradiated NSG mice.  Six weeks after engraftment, CG-SH sorted from the bone marrow of each individual mouse, along with pre-engrafted CG-SH, were subjected to next generation sequencing analysis using the TruSight Myeloid Sequencing Panel (Illumina, San Diego, CA) per manufacturers’ recommendations.  Briefly, the target oligo pool was hybridized with 100ng of high quality genomic DNA.  Extension and ligation of bound target oligos as performed followed by PCR amplification and PCR cleanup by AmpureXP purification  (Beckman Coulter, Brea, CA).  Library quantification was performed using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA), Qubit Flourometer (ThermoFisher, Waltham, MA), and KAPA qPCR (Kapa Biosystems, Wilmington, MA) and subsequent DNA sequencer performed with MiSeq v2 2x150 (Illumina, San Diego, CA ).  Variant analysis was performed using samtools version 1.0 "mpileup -q 5 -ugf " and bcftools version 1.0 "call -vmO v ". Annotation of the resulting SNP/INDEL calls was performed using annular (version date 2014-07-22) "table_annovar.pl --vcfinput --buildver hg19 --remove --protocol refGene,clinvar20140929,snp138 --operation g,f,f --nastring ."  Sequence variations in engrafted CG-SH from each mouse were compared to those in pre-engrafted CG-SH to identify new variations occurring after engraftment.