Myeloid DNA Reference Standard

The Myeloid DNA Reference Standard is a well characterized, cell line derived control material manufactured under ISO 13485 that contains 22 variants across 19 genes relevant to myeloid cancer. This exciting new Multigene Multiplex product allows labs to perform quick, more reliable and cost-effective validation of myeloid sequencing assays - saving the user time and money to achieve their quality assurance goals faster.

Please note that batch 54495 has been released with the NRAS Q61L variant at an allelic frequency of 12.41% instead of the normal 10%. All other variants passed QC testing within acceptance criteria. See Certificate of Analysis for all QC testing results. 

The Myeloid DNA Reference Standard contains a large number of variants within one tube –meaning users only need to handle one control sample in order to validate a large proportion of their targeted sequencing assay at once. This includes 6 insertions/deletions (INDELs), which can help users to optimize their assay’s performance when sequencing challenging structural variants. In particular, due to the importance of FLT3 internal tandem duplications (FLT3-ITDs), which are present in nearly 25% of patients with AML, Horizon has engineered a large novel 300bp insertion into FLT3 – which we have termed the “ITD300” variant. This is a very desirable feature of this new product, as it represents the types of large insertions that may be encountered in real patient samples but are very difficult to source for assay validation and optimization purposes. The Myeloid DNA Reference Standard is designed with 15 mutations present at 5% allele frequency, to support the calculation of clinically relevant LODs of somatic workflows. In addition, the product contains 7 mutations at 10-70% allele frequency to support the validation of higher frequency germline workflows.

 

Technical Data

Format: Genomic DNA

Genes Covered: ABL1, ASXL1, BCOR, CBL, DNMT3A, EZH2, FLT3, GATA1, GATA2, IDH1, IDH2, JAK2, KRAS NPM1, NRAS, RUNX1, SF3B1, TET2, TP53

Allelic Frequencies: 5 - 70%

Buffer: Tris-EDTA (10mM Tris-HCl, 1mM EDTA), pH 8.1

 

Product Information

Verified Mutations: The Myeloid Reference Standard has been manufactured using multiple engineered cell lines and mixed to generate multiple Allelic Frequencies in multiplex. An example of the allelic frequency by whole-exome sequencing for one batch of this product is reported in the table below.

Gene Variant (AA) CDS mutation GRCh38 co-ordinates Expected allelic frequency (%) NGS allelic frequency (%) NGS read depth
ABL1 T315I c.1001C>T 9:130872896 5.00% 5.23% 459
ASXL1 G646Wfs*12 c.1934dup 20:32434638 40.00% 39.66% 348
ASXL1 W796C c.2388G>T 20:32435100 5.00% 5.36% 522
BCOR Q1208Tfs*8 c.3621dup X:40063833 70.00% 68.44% 358
CBL S403F c.1208C>T 11:119278278 5.00% 5.62% 1334
DNMT3A R882C c.2644C>T 2:25234374 5.00% 6.26% 495
EZH2 R418Q c.1253G>A 7:148817379 5.00% 4.76% 714
FLT3 D835Y c.2503G>T 13:28018505 5.00% 4.71% 700
FLT3 ITD300 N/A 13:28033909 5.00% b b
GATA1 Q119* c.355C>T X:48791978 10.00% 8.05% 298
GATA2 G200Vfs*18 c.599del 3:128485998 35.00% 30.27% 403
IDH1 R132C c.394C>T 2:208248389 5.00% 4.42% 453
IDH2 R172K c.515G>A 15:90088606 5.00% 2.62% 535
JAK2 F537-K539>L c.1611_1616del 9:5070021 5.00% 4.01% 1147
JAK2 V617F c.1849G>T 9:5073770 5.00% 4.19% 1002
KRAS G13D c.38G>A 12:25245347 40.00% 36.04% 419
NPM1 W288Cfs*12 c.860_863dup 5:171410539 5.00% 3.89% 823
NRAS Q61L c.182A>T 1:114713908 10.00% 9.40% 628
RUNX1 M267I c.801G>A 21:34834414 35.00% 29.90% 194
SF3B1 G740E c.2219G>A 2:197401989 5.00% 3.14% 574
TET2 R1261H c.3782G>A 4:105243757 5.00% 4.94% 445
TP53 S241F c.722C>T 17:7674241 5.00% 5.82% 378

b – Variant type not called by variant caller (large insertion, CN variant, fusion, indel)

Unit Size: 500ng

Concentration: 25ng/µl

 

General Information

Storage: 4˚C

Expiry: See all product shelf life information

 

Quality Control

Allelic Frequency: Droplet Digital PCR™

Genotype: Sanger sequencing of locus specific PCR

Quality: Agarose gel electrophoresis

Quantification: Spectrophotometry (A260)