Open Access Highly Accessed Methodology article

New methods for next generation sequencing based microRNA expression profiling

Henk PJ Buermans1*, Yavuz Ariyurek2, Gertjan van Ommen1, Johan T den Dunnen12 and Peter AC 't Hoen1

Author Affiliations

1 Center for Human and Clinical Genetics, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands

2 Leiden Genome Technology Center, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands

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BMC Genomics 2010, 11:716  doi:10.1186/1471-2164-11-716

Published: 20 December 2010

Additional files

Additional file 1:

Figure S1. Realtime PCR validation of the miSeq libraries. Realtime PCR validation of the miSeq libraries prepared from heart tube and whole embryo RNA using the modified SREK protocol. Transcript specific forward primers for miR20b-5p, mir-206-3p and miR133 were based on full length miRbase [2] sequences and used in combination with a universal reverse primer that was designed to anneal to the 3' of the miSeq library as indicated in Figure 1A. miRNA expression was expressed relative to the total library content by using a forward primer at the 5' of the miSeq library.

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Additional file 2:

Table S1. EmiR expression table. smallRNA expression table as generated by the E-miR pipeline listing all expressed non-coding RNA transcripts from the annotation files that were detected in the sample data. Column description: The first column hold the transcript identifier, e.g., miRNA |ENSGALT00000028942|5p~|gga-mir-29ajsense, indicates the transcript is a miRNA with EnsemblTranscript ENSGALT00000028942 | the 5 prime part of the precursor j transcript name = gga-mir-29a and the match is the sense orientation. In cases where the miRbase has only one of the Dicer cleaved miRNA transcripts, the complementary transcripts were inferred from the hairpin structure. These unlisted transcripts are indicated by a '5p~' and '3p~' in the identifier. Expression for other non-coding RNA transcripts, like snoRNA and tRNA, are also included in the table. All of these have the '|n|' in the identifier instead of the |3/5p|. The second column holds the genome location. For each of the input files, seven columns of data are included, containing the following data: - unique: unique number of reads annotated to this miRNA transcript. - counts: sum of the number of times this miRNA transcript was found. - U0-counts: same as 'counts' but then only the sum of perfect matches only. - highest_count: expression of the most abundant isomer. - highest_seq: Identifier of the most abundant isomir: compiled from chr | begin | end | strand | mismatches in alignment | isomir sequence. - tpm scaled the 'counts' value normalized/scaled to sequences per million. - sqrt square root of the scaled value. This stabilizes variance.

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Additional file 3:

Figure S2. Embryo small vs total RNA scatter plot. Scatter plot indicating average miRNA expression in sqrt(tpm) for whole embryo derived libraries generated with small RNA enriched fractions and totalRNA. Open and closed black circles represent non-significant and significantly differentially expressed miRbase miRNA transcripts respectively. The top left insert depicts an enlarged section of the 0-20 sqrt(tpm) area. The table lists the FDR corrected p-value and expression levels in sqrt(tpm) for all six differentially expressed miRNAs.

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Additional file 4:

Figure S3. Short amplification primers. Alternative set of short primers used during miRNA sequencing library preparation to make the SREK protocol compatible with the Illumina Genome Analyzer. * Indicates a phosphorothioate bond.

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Additional file 5:

Table S2. Limma results. Significantly differentially expressed miRNAs between the Heart and Embryo from the Limma analysis. Columns 1 and 2 are in the same format as those in Additional file 2, Table S1, followed by the average expression levels, in sqrt(tpm), for the EMs and HTs library and BH-FDR p-value for the difference in expression.

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Additional file 6:

Figure S4. qPCR confirmation of sequencing data. MicroRNA expression levels for 5p or 3p transcripts in HH16 whole chicken embryo and heart tube. 5 s rRNA was used as an internal control to normalize gene expression. Left and right column represent miSeq and qPCR derived expression levels, respectively. The bars represent mean expression levels ± sd. indicates a significant difference in gene expression relative to whole embryo.

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Additional file 7:

Table S3. Globaltest results. Significantly differentially expressed miRNAs between the Heart and Embryo from the Globaltest analysis. Columns 1 and 2 are in the same format as those in Additional file 2, Table S1, followed by the BH-FDR p-value for the difference in expression and the number of isomirs the test was based upon. the last two columns indicate if the miRNA was significant in the Limma analysis and if the most abundant isomir had expression above 50 tpm to be included for further analysis.

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Additional file 8:

Methods S1. Modified smallRNA library preparation protocol. step-by-step protocol to generate microRNA sequencing libraries using the SREK kit that are compatible with the Illumina Genome Analyzer.

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Additional file 9:

Table S4. qPCR primers. miRNA specific forward primer sequences used for qPCR confirmation of sequencing data.

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