Additional file 1.
Flow chart of the procedure for sample preparation and sequencing, processing of reads and miRNA identification. (A) Flow chart of the procedure for sample preparation and sequencing. (1) P. tricornutum log phase cells were incubated in normal, nitrogen limited and silicon limited medium for 48 h and harvested, frozen instantly in liquid nitrogen and stored at -80°C before RNA extraction. (2) Total RNA was extracted using the Trizol method. (3) Fragments of 18-28 nt were gel-purified. (4) A 3' adaptor was ligated to the 3' end of sRNAs. (5) A 5' adaptor was ligated to the 5' end of sRNAs. (6) sRNAs were RT-PCR-amplified. (7) Sequencing. (B) Flow chart of the procedure for processing of reads. The numbers in parentheses represented the total reads from PT1, PT2 and PT3, respectively. (1) Initial processing: remove adapter, filter low quality tags and clean up tags smaller than 18nt. (2) Common/specific tags identified between samples. (3) Length distribution analysis of clean reads. (4) Matched clean reads to P. tricornutum nuclear genome using SOAP. (5) Matched clean reads to P. tricornutum chloroplast genome using SOAP. (6) Compared clean reads with non-coding RNAs from GenBank and Rfam. (7) Exon/intron fragment identified. (8) siRNA identified. (9) Plant miRNA homologs identified. (10) Annotated sRNAs. (11) Identified miRNA by hairpin structure filtering. (12) Target prediction. (C) Flow chart of the procedure for miRNA identification. (a) mfold was used to predict the secondary structure of extracted sequences. Sequences with Δ G < -18 kcal/mol, ≥ 16 bp and ≤ 4 bulges or asymmetries between miRNA and the other arm, miRNA sequence length between 18-25nt, with flank sequence length of 20, were obtained for further analysis. (b) randfold was used to check the stabilities of the candidate pre-miRNAs. (c) 5' homogeneity was checking. For precursors with a low P-value of ≤ 0.05 tested by randfold, a 5' homogeneity >0.5 was applied. For precursors with a P-value > 0.05, a 5' homogeneity ≥0.75 was applied. (d) Criteria made previously for miRNA identification were used to check the remaining sequences manually.
Format: JPEG Size: 2.8MB Download file
Huang et al. BMC Genomics 2011 12:337 doi:10.1186/1471-2164-12-337