Recently, a study was published that demonstrated an EG-based droplet digital PCR platform as well as a proof-of-concept multiplexing approach based on amplicon length with the QX200 system.
From the remaining loci, initial selection for inclusion was made based on amplicon length, number of alleles (high allelic diversity preferred), and inbreeding coefficient (loci with extreme deviation of Hardy Weinberg equilibrium were avoided).
For determining gene-specific LD estimates and extent of LD decay, the 32 SNP loci were identified in five seed and pod trait-associated TF genes among 96 chickpea genotypes based on cloned amplicon sequencing with an average SNP frequency of 1/297 bp.
Calculations were based on known amplicon size and concentration as determined with an Eppendorf BioPhotometer.
The gels were documented using VersaDoc Imaging System Model3000 (Bio-Rad Laboratories, Inc. USA) and allelic pattern was scored based on the amplicon size.
The consensus nucleotide at PpAG position 4757 was based on sequenced amplicons of three cultivars with melting profiles similar to 29 other cultivars.
The consensus nucleotide at PpTFL1 position 1202 was based on sequenced amplicons of nine cultivars with melting profiles similar to 24 other cultivars.
Product detection, allele calling, and hg inference were based on the amplicon-characteristic melting-point temperatures obtained with on-line post-PCR fluorescent dissociation curve analysis (DCA).
All 220 available homologous sequences of P. trichocarpa were identified in the JGI Database, based on 220 specific amplicons with SSR markers selected from P. tomentosa candidate gene sequences.
Thus in our comparisons of the functionality of conserved versus non-conserved sequences, we classified amplicons based on overlap with phastCons Placental Mammal Conserved Elements, 28-way Multiz Alignment intervals [ 41- 43].
