Carrot Genome Assembly V.2 RNAseq population 70796 dOr vs. pOr

This analysis was part of the carrot genome assembly publication

In population 97837, root tissue was collected from plants with yellow (yyY₂Y₂) and white (YYY₂Y₂) genotypes, with two biological replications per genotype, at 80 days after planting (DAP). In population 70796, root tissue was collected from plants with dark orange (yyy₂y₂) and pale orange (YYy₂y₂) genotypes, with three biological replications per genotype, at 100 DAP. Total RNA was extracted from whole root tissue using the TRIzol® Plus RNA Purification Kit (Life Technologies, Carlsbad, CA) in accordance with the manufacturer’s protocol. RNA was treated for DNA contamination with the TurboDNA-free kit (Life Technologies, Carlsbad, CA). RNA quantity and integrity was confirmed with an Experion RNA StdSens Analysis kit (Bio-Rad, Hercules, CA). All samples had RQI values above 8.0.

For each biological replicate, a 133 nt insert size paired-end library was prepared at the Biotechnology Center, UW-Madison (WI, USA). Libraries were sequenced on Illumina HiSeq2000 lanes using 2 × 100 nt reads. Reads were filtered using Trimmomatic version 0.32 with adapter trimming and using a sliding window of length ≥50 and quality ≥28, i.e. “ILLUMINACLIP:adapterfna:2:40:15 LEADING:28 TRAILING:28 SLIDINGWINDOW:10:28 MINLEN:50”.

Filtered reads were aligned to the Daucus carota v2.0 genome assembly using the program TopHat v2.0.12 (ref. 30). Non-default parameters used were “--mate-inner-dist -67 --mate-std-dev 50 --min-intron-length 20 --max-intron-length 10000 --library-type fr-unstranded --num-threads 14”. The aligned read files were processed by Cufflinks v2.2.1 (ref. 61). Reads were assembled into transcripts with “cufflinks” using the carrot annotation v1.0 gene predictions as the reference gtf guide. Samples were combined with “cuffmerge”, and then differential expression analyzed with “cuffdiff”, using non-default parameters of “--multi-read-correct --min-alignment-count=5”. Using the abundance estimations, this performs tests for differential expression and regulation between the samples. Normalized counts of the mapped RNA sequences were used to calculate the relative abundances of transcripts expressed as Fragments Per Kilobase of exon per Million fragments mapped (FPKM). When testing for differential expression, biological replicates were included as a term in the mixed model analysis to account for experimental error. Testing for differential expression was done at the level of genes, isoforms, and promoters.

Some of the data from this analysis were published in
Sup. Table 44: Annotation of carrot genes upregulated in both yellow and dark orange (yy) storage roots of plants from mapping populations
and in
Sup. Table 45: Annotation of carrot genes downregulated in yellow or dark orange (yy) storage roots of plants from mapping populations
of the carrot genome assembly publication