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NEBNext Direct® Genotyping Solution

The NEBNext Direct Genotyping Solution has been developed to provide a cost-effective, scalable solution to address the needs of genotyping for applications including marker-assisted breeding in plants. The approach is capable of supporting high-throughput processing of samples through high levels of sample multiplexing prior to hybridization-based enrichment of marker loci. In this method, individual DNA samples are fragmented and indexed-tagged in 96-well plates. Up to 96 samples are subsequently pooled together into a single tube for hybridization-based capture using biotinylated baits for 90 minutes. Following hybridization, the target fragments are bound to streptavidin beads and trimmed to remove flanking 3′ off-target sequence, sequencing adaptors are ligated, and the captured fragments are PCR amplified to generate full length libraries. 

Data available for marker sets in:

  • Rice (O. sativa): 1996 markers
  • Tomato (S. lycopersicum): 2300 markers
  • Maize (Z. maize): 4600 markers
 

Figure 1: NEBNext Direct Genotyping Solution workflow 





Figure 2: Passing filter reads across 96 pooled samples



Passing filter reads across 96 tomato DNA samples that were enriched using a genotyping panel consisting of 2,309 publicly available SolCAP markers and the NEBNext Direct Genotyping Solution. 25 ng of purified tomato DNA was used for each sample. Samples were index-tagged and pooled prior to hybridization and libraries were sequenced on an Illumina® MiSeq® with 20 cycles of Read 1 to sequence the 12 base UMI and 8 base sample index, and 75 cycles of Read 2 to sequence the targets.


Figure 3: NEBNext Direct Genotyping Solution demonstrates similar coverage across 96 pooled samples



Mean SNP coverage of 2,309 SolCAP markers across 96 samples. 25 ng of purified tomato DNA was used for each sample. Samples were index-tagged and pooled prior to hybridization and Libraries were sequenced on an Illumina MiSeq with 20 cycles of Read 1 to sequence the 12 base UMI and 8 base sample index, and 75 cycles of Read 2 to sequence the targets.


Figure 4: Specificity of enrichment of NEBNext Direct Genotyping Solution across 96 pooled samples



The percent of passing filter reads mapping to targeted regions demonstrates high specificity across 96 multiplexed samples using the NEBNext Direct Genotyping Solution. 25 ng of purified tomato DNA was used as input for each sample. Samples were index-tagged and pooled prior to hybridization and Libraries were sequenced on an Illumina MiSeq with 20 cycles of Read 1 to sequence the 12 base UMI and 8 base sample index, and 75 cycles of Read 2 to sequence the targets.


Figure 5: Mean Coverage across 2309 markers within a single sample



Histogram of coverage across each of the 2,309 SolCAP markers demonstrates evenness of enrichment across targets and coverage levels sufficient for genotyping calls. These data represent enrichment of a single tomato sample pooled with 95 others prior to hybridization. 25 ng of purified tomato DNA was used for each sample. Samples were index-tagged and pooled prior to hybridization and libraries were sequenced on an Illumina MiSeq with 20 cycles of Read 1 to sequence the 12 base UMI and 8 base sample index, and 75 cycles of Read 2 to sequence the targets.


Advantages

  • Processing of up to 9,216 samples in a single 96-well plate
  • Pre-capture pooling up to 96 samples
  • Single tube capture of 100-5,000 markers
  • Compatible with variety of extraction protocols
  • Single-day sample to sequencer workflow

 

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Genotyping Solution?

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