Iso-Seq analysis of barley CI 16151 and fast-neutron-derived, immune-compromised mutants infected with the powdery mildew fungus (Blumeria graminis f. sp. hordei; isolate 5874)

Purpose: The powdery mildew fungus, Blumeria graminis, is an obligate biotrophic pathogen of cereals and has significant impact on food security (Dean et al., 2012. Molecular Plant Pathology 13 (4): 414-430. DOI: 10.1111/j.1364-3703.2011.00783.x). Blumeria graminis f. sp. hordei (Bgh) is the causal agent of powdery mildew on barley (Hordeum vulgare L.). We sought to discover novel transcripts expressed following barley infection with blumeria. Overall design: 90 pooled samples analyzed = 5 genotypes * 6 time points * 3 replications. The pooled sample (90 experimental units) was SAGE-ELF size selected to generate 6 PacBio cDNA Iso-Seq Library Preparations [fragment sizes: 1) non-fractionated 2) 1000-1500 nt 3) 1500-2000 nt 4) 2000-3000 nt 5) 3000-5000 nt 6) > 5000 nt]. The non-fractionated as well as the three smallest fragment libraries were each loaded onto 3 SMRT cells, and the two largest fragment libraries were each loaded onto 2 SMRT cells for a total of 16 SMRT cells. Four error-correcting programs (HECIL, LorDEC, HALC, COLORMAP) were explored to fix indels in the long PacBio reads. Each of these programs aligns short-reads directly to the erroneous PacBio long reads and uses the pile-up to correct insertions, deletions, and mismatches. Error-corrected long reads were mapped back to the Morex genome and the number of mismatches and indels were recorded. For each long read, the software that produced the best mapping quality was retained. Note: This experiment used the identical split-plot design, tissue, and source RNA as GEO submission # GSE101304 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE101304). Gene counts were also obtained from short-reads generated from GEO submission # 101304 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE101304). These transcripts will be used as a reference gold standard annotation to compare results from different gene annotation pipelines. The short reads were not re-aligned to the new fasta files.