PG-Seq™ Rapid kit v2 assessment summary
4Pages

TECHNICAL NOTE PG-Seq™ Rapid kit v2 assessment summary. -\r Authors Kimberly Warren Revvity, Inc. For research use only. Not for use in diagnostic procedures. Introduction PG-Seq™ Rapid kit v2 was developed to offer a streamlined workflow and improved whole genome coverage to facilitate an improved user experience and allow compatibility for optional downstream applications. Methods Cell lines of the following karyotypes; 47,XX,+18 (GM00143), 47,XY,+15 (GM07189), 48,XXY,+21 (GM04965) and genomic DNA 48,XY,+2,+21 (GM03576), 47,XXX (NA04626), 46,XX,del(13) (pter>q14.1::q21.2>qter) (NA07312) and 47,XY,+der(21) t(3;21)(p24.1;q21) (NA09552) were purchased from Coriell Biorepository (USA). Peripheral lymphocytes from a male were also isolated from peripheral blood mononuclear cells and were designated 46,XY. Cells were washed in droplets of PBS and manually grouped together in 5-cell aliquots before being transferred to a PCR tube and stored at -20°C. Genomic DNA was quantified and serially diluted in 10mM Tris-HCl pH 8 to a final concentration of 30pg/pL Cell lysis, WGA PCR 1 and Indexing PCR 2 was performed using 5-cell samples and 30pg dilutions of genomic DNA, according to the instructions in the PG-Seq™ Rapid kit v2 user manual. Following Cell lysis and WGA PCR 1, the samples were visualized via LabChip® GX II Touch™ HT nucleic acid analyzer (figure 1) to determine WGA success. After Indexing PCR 2, samples underwent the clean-up and size selection protocol, either individually or as a pool of samples. Final library pools were analyzed for fragment size on the LabChip® GX II

PG-Seq™ Rapid kit v2 assessment summary. Touch™ HT nucleic acid analyzer (figure 2). Sequencing was application on Basespace (Illumina, USA) and the resultant performed on an Illumina® MiSeq® instrument or Illumina® BAM files were down-sampled to 400,000 reads before MiniSeq® instrument with 1x75bp single index reads, analysis with the PG-Find™ v3 software. The PG-Find™ self- aiming for approximately 500,000 reads per sample or the reference algorithm was utilized with the default settings, equivalent of 48 samples per run on the Illumina® MiSeq® and one adjustment of the significance...

PG-Seq™ Rapid kit v2 assessment summary. Figure 6b: 30pg genomic DNA sample GM07312, 46,XX,del(13) (pter>q14.1::q21.2>qter), closer view of the 18Mb loss on chromosome 13 Figure 7b: 30pg genomic DNA sample NA09552 47,XY,+der(21) t(3;21) (p24.1;q21), closer view of the 31Mb gain on chromosome 3.

PG-Seq™ Rapid kit v2 assessment summary. Figure 8: 30pg genomic DNA sample NA03576 48,XY,+2,+21 Conclusions The PG-Seq™ Rapid kit v2 offers analysis of whole chromosome and sub chromosomal copy number changes down to 7 Mb in size. The data presented in this study was generated with 5-cell aliquots and diluted genomic DNA extracted from cell lines with known karyotypes as a model of trophectoderm biopsy. The amplification is reliable, flexible, easy to use and in this study showed 100% amplification success and 98% of samples generating a result that passed quality control metrics. Correct...