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Pipeline publication

[…] 4°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 2 min; then extension of products at 72°C for 10 min. The PCR products were separated by electrophoresis on 8.0% non-denaturing polyacrylamide gels, silver-stained, and band sizes assessed by comparison to a DNA ladder., cDNA was prepared from 5 days, 15 days, and 30 days fruits after flowering and sequenced with Illumina HiSeq 4000 platform. A total of 46,486,152, 49,649,472, 56,409,052 raw reads were generated, after stringent quality assessment and data filtering, a total of 44,190,154, 47,458,054, 53,607,998 clean reads were generated for 5 days, 15 days, and 30 days fruits. All high-quality reads were assembled using trinity software [], yielding a total of 219,831 transcripts with average length of 771 bp and N50 of 1302 bp (). The length distribution of transcripts is showing in . The de novo assembled transcriptomes were clustered by ‘corset’, which is a method and software for obtaining gene-level counts from any de novo transcriptome assembly []. After clustering by corset, a total of 139,863 clusters were obtained, in which the clusters with longest sequences were defined as unigenes. Finally a total of 139,333 unigenes with average length of 1049 bp and N50 of 1579 bp () were obtained from the transcripts. The length distribution of unigenes is showing in . The length distribution comparison of transcripts and unigenes is showing in ., To validate the assembly quality and annotation of the assembled unigenes, all unigenes were used to seek matches in public databases including Nr, Nt, Ko, Swiss-prot, Pfam, GO and KOG/COG using the BLASTx program with an E-value threshold of 1E-5. Among 139,333 unigenes, a total of 12,246 (8.78%) unigenes were annotated in all databases, and 92,498 (66.38%) matched genes and/or proteins in at least one database. The detailed results are shown in and ., Based on the Nr database, of the assembled sequences, 66.76% showed significant homology (<1E-50), and 70.09% showed more than 80% similarity to Nr database entries (). The L. barbarum unigenes were homologous to sequences in other species, among which Solanum tuberosum accounted for 30.2% (19,388), Nicotiana sylvestris accounted for 20.4% (13138), Nicotiana tomentosiformis accounted for 19.9% (12,793), Solanum lycopersicum accounted […]

Pipeline specifications

Software tools Trinity, Corset, BLASTX
Chemicals Hydrocarbons, Acyclic