Fig 1.
Schematic diagram of the TeloPrime and SMARTer cDNA synthesis technologies.
(A) SMARTer does not distinguish between truncated and full-length transcripts. (B) TeloPrime enriches for full-length transcripts by affinity of the 5’ adapter to the 5’ cap structure of full-length mRNAs during cDNA synthesis. Red and pink rectangular shapes are the 5’ adapters provided by each kit, respectively. Green rectangular boxes are the oligos priming cDNA synthesis of poly-A transcripts.
Table 1.
Arabidopsis thaliana PacBio Iso-Seq output summary.
cDNA was synthesised with Lexogen TeloPrime Full-Length amplification kit or the Clontech SMARTer PCR cDNA Synthesis Kit and then split into three size fractions (1–2, 2–3 and 3–6 kb), respectively. From left to right: number of transcript isoform clusters (HQ reads) assembled using the PacBio Iso-Seq pipeline; number of HQ reads aligning to the A. thaliana genome with a sequence identity > = 90%; percentage of full length HQ reads (FL) defined as the cumulative number of reads equal or larger in length than their respective target.
Fig 2.
Transcription start and end site enrichment using the TeloPrime Full-Length cDNA amplification kit (Lexogen).
(A-C) Superimposed density plots of the PacBio Iso-Seq alignment coordinates against the 5’ and 3’ ends of their targets. TeloPrime cDNA libraries (red bars), SMARTer cDNA libraries (light blue bars), overlay of the two protocols (brown bars). (A) 1–2 kb size fraction; (B) 2–3 kb size fraction; (C) 3–6 kb size fraction. 10 kb around the annotated gene start and end coordinates are shown on the x-axis. Vertical green dashed lines highlight annotated target start and end sites.
Fig 3.
Improved resolution of transcription start and end sites in T. aestivum.
Density plot of PacBio full-length cDNA read alignment ends within 10 kb around the target start and end coordinates are shown for each size fraction; (A) 1–2 kb size fraction; (B) 2–3 kb size fraction; (C) 3–6 kb size fraction. Vertical green dashed lines highlight target start and end sites.
Table 2.
Genomic regions detected by the wheat HQ reads.
The number of HQ reads aligning to each genome and scaffold. The last column indicates the number of full length reads (FL, i.e. equal or larger in size than the corresponding target) calculated for long and small coding genes and pseudo-genes. Pt: chloroplast.
Fig 4.
Best blast hit distribution against the nr database using HQ reads not aligning to the T. aestivum Ensembl genome assembly.