Supplementary Figures and Legends

Supplementary Figure 1. Detailed TIF-Seq protocol. (1) Non-capped RNA molecules are dephosphorylated. (2) Using the oligo-capping method28 a known oligo (orange) is ligated to the capped mRNA molecules. (3) Sample is split and barcoded full-length cDNA is produced by reverse transcription and PCR amplification. (4) Sticky ends are produced by NotI digestion; samples are pooled and circularized by intermolecular ligation. (5) Circularized molecules are purified and fragmented by sonication. 5'-3' junctions are captured by biotin-streptoavidin purification. (6) Illumina libraries are produced and sequenced using 105-nucleotide paired-end reads.

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SUPPLEMENTARY INFORMATIONdoi:10.1038/nature12121

Supplementary Figure 2. Clustering of transcript boundary positions for the definition of major transcript isoforms (mTIFs). a, Criteria for mTIF clustering. When comparing distances between TSSs from all overlapped TIFs, start sites separated by 8 nucleotides (red line) are overrepresented. A 5 nucleotide clustering distance for mTIFs was chosen to stay well above our technical precision while maintaining the resolution necessary to identify the 8 nucleotide-spaced start sites, which could have a biological basis. In the following panels, individual transcription start sites (purple) or polyadenylation sites (orange) are displayed in natural and logarithmic scale; sites co-occurring within 5 nucleotides were clustered into mTIFs (dark blue boxes, Supplementary Methods). Individual TIFs are displayed as blue lines, coverage as dark blue. b, Example of small boundary variations with large functional consequences: 5' end variations of only a few nucleotides in HTS1 determine whether or not the 1st AUG is included and thus whether the transcript encodes the mitochondrial or cytoplasmic version of histidyl-tRNA synthetase respectively29, as the former requires an N-terminal signal peptide (also shown in Supplementary Fig. 20 with other examples of isoforms encoding truncated proteins). c, The same clustering was applied to 3' ends, illustrated here for ALT1; the logarithmic plot demonstrates the prevalence of isoforms with minor variations that would have been discarded with a larger window for clustering. d, In some cases transcription start sites can also span hundreds of nucleotides, shown here for ALT1. Protein coding sequences (in b and d) are represented as in Fig. 4a.

319976 320049 320080 320110 320129 320155 320183 320210 320236 320281 320313 320334 320358 320373 320398 320413 320439

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5 Start (-) Logarithmic scale [0-875]

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mTIF 5 Start clusters

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638947 638966 638975 638987 638998 639010 639020 639031 639051 639064 639090

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G C A T T A G C A G T A G G A G C A G A A G T A G C A G C A G C G G T A G C G G A T G A C A T C C T G A T T A T C G A A G A C G A C T T T A T A G A T G T G A C T A C T T T A T T T A G T G A T C T A C T A A G C A T T A A G C A A G A G A C G A G C A G A A A G G A A C T A T T C T A A A G T A A C A C A T T G G G C C G T A A G A C T C A T T T A A A A G C C C G G T C G A T C

A N A T P A S T A A A T A S S M R I I S S S K I S T V V K N L S R S L M

Chr XVI 638,940 bp 638,960 bp 638,980 bp 639,000 bp 639,020 bp 639,040 bp 639,060 bp 639,080 bp

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SUPPLEMENTARY INFORMATIONRESEARCHdoi:10.1038/nature12121

Supplementary Figure 3. Reproducibility of the TIF-Seq method. Correlation between technical replicates of chimaera control configurations (A-A) and (B-B) of one sample in YPD (YPD1, see Supplementary text); and biological replicates YPD1 and YPD2. Spearman correlation values (cor) are shown. a, Correlation between the numbers of exactly identical TIFs (identical start and end sites) in A-A and B-B; b, Correlation between the numbers of TIFs overlapping with annotated transcripts in A-A and B-B; c, Correlation between the numbers of exactly identical TIFs (identical start and end sites) in biological replicates YPD1 and YPD2; and d, Correlation between the numbers of TIFs overlapping with annotated transcripts4 in YPD1 and YPD2.

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