nextera-based amplicon sequencing  (Nextera AS)

Journal: G3: Genes|Genomes|Genetics

Article Title: Challenges and Approaches to Genotyping Repetitive DNA

doi: 10.1534/g3.119.400771

Figure Lengend Snippet: (A) Schematic of the C. elegans rDNA locus, at the right arm of chromosome I. The 18S, 5.8S, and 26S rRNAs are transcribed as one unit and later processed into the three species. One repeat unit is 7.2kb in length and is tandemly repeated approximately 70 to 400 times, depending on the strain. The array is flanked by a partial copy of the 26S rRNA gene ( rrn-3.56 ) and an additional copy of the 18S ( rrn-1.2 ), which ends approximately 1kb upstream of the telomere. (B) Southern blot against rDNA from a CHEF (contour-clamped homogenous electric field) gel reveals rDNA copy number for eight wild isolates of C. elegans . Band size was measured relative to yeast chromosomal ladders visualized by ethidium bromide staining (Fig. S1), and copy numbers calculated from the band size for each primary band are listed in red (also in ). MY16 displays two bands of similar intensity, copy numbers 83 and 69; only the upper number is listed in the image. (C) rDNA copy number (per haploid genome) was estimated for eight C. elegans wild isolates in five different ways: CHEF gel followed by Southern blot, 10ng input Nextera-based whole genome sequencing followed by relative read count coverage of the rDNA without or with GC content correction (“RR” and “GCC,” respectively), and 50ng input whole genome sequencing with RR and GCC. Three replicates for each are plotted. (D) Data from (C) is plotted as a percentage of the average CHEF-based values ((estimated number – CHEF number)/CHEF number)). The red line indicates 0% error.

Article Snippet: We evaluate pulsed-field gel electrophoresis, droplet digital PCR, and Nextera-based whole genome sequencing as approaches to copy number estimation, comparing techniques across model organisms and spanning wide ranges of copy numbers.

Techniques: Southern Blot, Staining, Sequencing

Journal: G3: Genes|Genomes|Genetics

Article Title: Challenges and Approaches to Genotyping Repetitive DNA

doi: 10.1534/g3.119.400771

Figure Lengend Snippet: rDNA copy number estimates of C. elegans strains using CHEF gel or WGS

Article Snippet: We evaluate pulsed-field gel electrophoresis, droplet digital PCR, and Nextera-based whole genome sequencing as approaches to copy number estimation, comparing techniques across model organisms and spanning wide ranges of copy numbers.

Techniques: Sequencing

Journal: G3: Genes|Genomes|Genetics

Article Title: Challenges and Approaches to Genotyping Repetitive DNA

doi: 10.1534/g3.119.400771

Figure Lengend Snippet: (A) Schematic of the S. cerevisiae rDNA locus on chromosome XII. The 18S, 5.8S, and 25S are transcribed as one unit by PolI. 5S rRNA is transcribed as a separate unit by PolIII. The repeating unit is 9.1kb in length and in haploid strains is tandemly repeated approximately 90 to 300 times. (B) Southern blot for chromosome XII from a CHEF gel reveals size variation of S. cerevisiae strains from the 1002 genomes project (Peter et al . 2018). rDNA copy numbers measured from each band are noted in red, calculated from band size relative to yeast chromosomal ladders. Haploid and diploid strains are noted. Wild haploid yeast strains show copy numbers ranging from 91-306. Wild diploid strains show individual bands with copy numbers ranging from 45-292 (note that two strains, AFC and AAC had bands at the lower limit of our ability to quantify, estimated as 5 and 1 copies, respectively). From left to right, strains were arranged in order of the expected rDNA copy number, based on previously reported whole genome sequencing estimates. (C) rDNA copy numbers for 30 strains of S. cerevisiae isolates are plotted, reflecting CHEF-based rDNA copy number estimates(“CHEF”, dark blue), previously reported data (“SRA data”, green), or our own Nextera whole genome sequencing-based estimate (“Re-sequenced”, mauve, done for 14 strains plus B30 and BY4741 controls). The annotation “err” indicates strains whose re-sequenced genotype did not match SRA library genotypes. The SRA-based estimate has therefore been omitted from the graph for these strains. (D) Droplet digital PCR estimations (in triplicate) of rDNA copy number for eight yeast strains are plotted next to their CHEF-based rDNA copy numbers.

Article Snippet: We evaluate pulsed-field gel electrophoresis, droplet digital PCR, and Nextera-based whole genome sequencing as approaches to copy number estimation, comparing techniques across model organisms and spanning wide ranges of copy numbers.

Techniques: Southern Blot, Sequencing, Digital PCR

Journal: G3: Genes|Genomes|Genetics

Article Title: Challenges and Approaches to Genotyping Repetitive DNA

doi: 10.1534/g3.119.400771

Figure Lengend Snippet: (A) Single molecule Molecular Inversion Probes (smMIPs) were targeted against regions of the rDNA (blue) and against four different single copy loci in the genome (green). A normalization plasmid contained a single copy of the rDNA repeat unit as well as the sequences of the four single copy loci, all with unique nucleotide variants (orange lines), only the two most consistent of which were used in the final analysis. smMIPs capture ∼100-150bp sections of the target DNA, with each capture event individually barcoded. Single-read sequencing was used to identify the barcode of each smMIP and source of its captured DNA (plasmid or genomic). The number of plasmids per genome was calculated from the ratio of genomic single copy loci smMIP capture events to normalization plasmid capture events. The rDNA copy number was estimated from genomic rDNA capture events relative to plasmid rDNA capture events, corrected for number of plasmids per genome. In this way, variable efficiency of probe binding is normalized. (B) Molecular inversion probe-based copy number estimation was performed using different input DNA amounts: 5ng and 10ng. rDNA copy number estimations are plotted here for eight wild C. elegans strains, alongside their CHEF-based copy numbers (dark blue). (C) 5ng and 10ng input amounts are compared against the CHEF-based rDNA copy number for the eight strains. Data are plotted as a percentage of the average CHEF-based values ((estimated number – CHEF number)/CHEF number).

Article Snippet: We evaluate pulsed-field gel electrophoresis, droplet digital PCR, and Nextera-based whole genome sequencing as approaches to copy number estimation, comparing techniques across model organisms and spanning wide ranges of copy numbers.

Techniques: Plasmid Preparation, Sequencing, Binding Assay