Amplicon sequencing of Homo sapiens: VDJ regions within B-ALL
Genomic DNA (gDNA) was isolated (Wizard Genomic DNA Purification; Promega) from frozen cell pellets, unsorted viably frozen samples, a paraffin-embedded skin biopsy, and from cells sterilely extracted from CSF cytospin slides with a Surgical Scalpel Blade No.11. DNA was quantified by absorbance at 260 nm (NanoDrop, Thermo Fisher Scientific) and dsDNA binding of fluorescent dyes (Qubit, Thermo Fisher Scientific). PCR amplification was performed using Q5 High Fidelity Polymerase (New England Biolabs, Inc., Ipswich, MA) using up to a maximum of 500 ng genomic DNA as template divided across 4 amplicon reactions. For CSF specimens without 500 ng gDNA available, all available DNA was likewise divided across 4 amplicon reactions. Recombined IgH primers were specific to each joining (J) gene (J1-6) and family specific Framework 2 (FR2) variable (V) gene of the IgH locus (Table S1). An ultraconserved region (eUCR41) found on Chr 11 was also amplified for use as an internal control for gDNA preparation damage, PCR, and NGS errors2. All amplicons were of similar size of approximately 250-300 nucleotides. Gene-specific primers were modified by the addition of NexteraXT sequences (Illumina Inc., San Diego, CA) at the 5 primer end. Amplicons were size-selected and purified (SPRIselect; Beckman Coulter) to maximize selection of 300 nt amplicons for NGS. Specimens were sequenced on an Illumina MiSeq System using 2x300 bp sequencing chemistry. Library preparation and sequencing was performed in the Genomics Research Center, University of Rochester, Rochester, NY.