- Complete BRCA1 and BRCA2 coverage
- 100% uniformity of all targeted coding exons and exon-intron boundaries
- Shortest Illumina® targeted library prep protocol available - 2 hour protocol validated with low input samples
- For detection of germline mutations multiplex up to 384 samples at 100x coverage and for detection of somatic mutations multiplex 14 samples at 2000x coverage on a single Illumina® 2x300 MiSeq® lane
- Complete solution for targeted sequencing including gene specific primers, PCR Master Mix, clean-up beads and up to 384 barcodes
- Low input - only 20 ng DNA required from fresh or frozen tissue or blood
For Research Use Only. Not for use in diagnostic procedures.
Complete BRCA1 and BRCA2 Coverage for Illumina® Sequencing
The NEXTflex® BRCA1 & BRCA2 Amplicon Panel allows you to quickly target and sequence all exons of the BRCA1 and BRCA2 loci. This kit contains primer pairs and reagents needed to amplify the coding regions of BRCA1 and BRCA2 and prepare libraries for Illumina sequencing.
A Complete Solution for Targeted BRCA1/2 Sequencing
The NEXTflex BRCA1 & BRCA2 Amplicon Panel is a complete, validated solution that targets all exons in BRCA1 and BRCA2 loci using 130 PCR primer pairs. This kit requires only 20 ng of DNA from fresh or frozen samples. This kit is not designed for use with DNA isolated from FFPE or cfDNA samples. The convenient predesigned panel allows researchers to focus on data generation and analysis, not on labor-intensive primer design and target selection. While competitor protocols typically require several hours to perform, the NEXTflex BRCA1 & BRCA2 Amplicon Panel produces targeted Illumina® libraries in only 2 hours. Additionally, the NEXTflex BRCA1 & BRCA2 Amplicon Panel protocol can be easily automated on liquid handlers, further simplifying workflow.
The NEXTflex® BRCA 1 & BRCA 2 Amplicon Panel contains enough material to prepare 8, 48 or 96 samples for Illumina® sequencing from DNA isolated from fresh or frozen tissue. The shelf life of all reagents is 12 months when stored properly. All components can be safely stored at -20°C.
NEXTflex® BRCA1/2 Amplicon Panel Kit Contents
NEXTflex® BRCA Amplicon Primer Mix 1
NEXTflex® BRCA Amplicon Primer Mix 2
NEXTflex® PCR II Barcoded Primer Mix
NEXTflex® PCR Master Mix
NEXTflex® Cleanup Beads
Required Materials Not Provided
Two aliquots of 10-50 ng of high-quality genomic DNA in up to 26.4 µL nuclease-free water each
96 well PCR Plate Non-skirted (Phenix Research®, Cat # MPS-499) or similar
Adhesive PCR Plate Seal (Bio-Rad®, Cat # MSB1001)
Magnetic Stand -96 (Thermo Fisher Scientific®, Cat # AM10027) or similar
2, 10, 20, 200 and 1000 μL pipettes / multichannel pipettes
Nuclease-free barrier pipette tips
80% Ethanol, freshly prepared (room temperature)
Recommendations for the Analysis of Sequencing Data
We recommend paired-end sequencing on an Illumina® MiSeq® with 2 x 300 bp reads (v3 cartridge) or 2 x 150 or 2 x 250 bp reads (v2 cartridge). If the full capacity of the cartridge can be used, then 2 x 300 bp v3 cartridges is more economical option than 2 x 150 or 2 x 250 bp v2 cartridges because, while higher priced, 2 x 300 bp v3 cartridges provide the option to sequence twice as many reads compared to v2.
While the Illumina® MiSeq® software provides an adapter sequence masking option, we recommend using a dedicated trimmer software, to ensure complete 3' adapter removal. We recommend the Trimmomatic. This software is best used in paired end mode. The minimum length of reads not removed after adapter trimming should be set to 69 (which is the length of the longest possible primer dimer).
Primer Sequence Removal
Primer sequences, which do not provide useful sequence information, can be removed from trimmed reads before the alignment. We suggest the use of Cutadapt software in paired 5' anchored adapter removal mode. This adapter removal mode will remove the 5’ primer sequences of all amplicons provided with the kit from both R1 and R2 reads. The primer fasta file should be modified by inclusion of a caret sign (^) at the 5' end of each primer sequence. 3' primer sequences which could be more difficult to unequivocally identify can be removed by instructing Cutadapt to remove 35 bp (which is the length of the longest primer) from the 3' end of each read. For 2 x 150 sequence results, do not remove the 3’ primer sequence.
Bowtie2 aligner in end-to-end alignment mode and other commonly used aligners (e.g. BWA) may be used. Using the paired reads alignment option is recommended. The BEDfiles provided are for the hg38 version of human genome.
For mutation calling, SAM tools mpileup can be used, followed by bcftools, to produce binary bcf files which are then converted to vcf. Alternatively GATK Unified Genotyper of the GATK pipeline could be used.