Journal: PeerJ

Article Title: Identifying transcript 5′ capped ends in Plasmodium falciparum

doi: 10.7717/peerj.11983

Figure Lengend Snippet: Plasmodium falciparum mRNA was spiked with SIRV set 3 (a pool of synthetic RNAs with known 5′ capped ends). Two experiments were conducted using 5′ adapter ligation method1 (M1_1 and M1_2) and two with 5′ adapter ligation method2 (M2_1 and M2_2). To test whether the power to detect was increased by combining enrichment P -values from different experiments, combined P -values were calculated from M1_1 and M1_2 experiments (M1_combined), M2_1 and M2_2 experiments (M2_combined) and all four experiments (all_combined). Enrichment P -values were transformed to integer counts and clusters of enrichment signals were identified by analysis with CAGEr . CAGEr cluster signals were used for further analyses. (A) Example of transcriptomic data for the ERCC-00002 synthetic RNA. Reads from enriched and unenriched libraries were aligned to the reference sequence (SIRvomeERCCome). Data tracks underneath the chromosome line show normalized read depth for each library, with the range of depth values in the interval shown indicated in brackets. The bottom data track shows the integer counts of 5′ capped nucleotide enrichment from four experiments (5′ cap enrichment) with the range of integer counts in the interval shown indicated in the bracket. Figure produced using IGV software . (B) Receiver Operator Characteristic (ROC) plots. The black circle on the “all_combined” plot represents the optimal cut-point of enrichment signal (CAGEr cluster tag sum) equal to 32. (C) Empirical cumulative distribution frequency plots of natural log RNA concentration of External RNA Controls Consortium (ERCC) synthetic RNAs. The ERCC RNAs are the subset of synthetic RNAs with a single transcript annotated for each in silico gene. The 5′ end was detected for 34 ERCC RNAs, whereas the 5′ end was not detected for 58 ERCC RNAs. The distributions of the two groups were compared by two-sample Kolmogorov–Smirnov test ( P -value = 1.142e −07 ). (D) Correlation of natural log (CAGEr cluster tag sums) with natural log concentration of ERCC synthetic RNAs with detected 5′ end. The red line represents linear regression of the data, P = 3.941e −08 . 95% confidence intervals are indicated by the gray bands.

Article Snippet: The empirical cumulative distribution functions of detected and undetected 5′ ends with respect to natural log of ERCC RNA concentration reported by the manufacturer (Lexogen) were determined using the base R ecdf function.

Techniques: Adapter Ligation, Transformation Assay, Sequencing, Produced, Software, Concentration Assay, In Silico