Supplemental information. Supplementary_Fig_S1: Ribo-seq meta profiles at start and stop codons S. Typhimurium.

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1 Supplementl informtion Pge 1 Supplementry_Fig_S1: Rio-seq met profiles t strt nd stop odons S. Typhimurium. 2 Supplementry_Fig_S2: Red length distriutions t Shine Dlgrno motifs. 3 Supplementry_Fig_S3: Codon speifi red length distriutions. 4 Supplementry_Fig_S4: Additionl predition support. 4 Supplementry_Fig_S5: Third odon periodiity nd GC ontent. 5 Supplementry_Fig_S6: Evidene for predited novel trnsltion initition sites. 5 Supplementry_Fig_S7: Rio-seq met profiles t strt odons E. oli. 6 Supplementry_Fig_S8: Lirry red length distriutions. 7 Supplementry_Fig_S9: Red length djustments. 7

2 d Supplementry_Fig_S1: Rio-seq met profiles t strt nd stop odons - S. Typhimurium Rio-seq met-profiles in windows round strt odons for nnotted genes (n=4205) in the S. Typhimurium genome, ontriutions from eh gene re sled to sum of one. (upper) Proportion of 5 or 3' rio-seq red ounts per nuleotide position, oloured y odon position. (lower) Hetmps of 5' or 3' rio-seq red ounts per length, oloured y z-sore per proteted red length. 2

3 Supplementry_Fig_S2: Red length distriutions t Shine Dlgrno motifs Rio-seq met-profiles in windows round shine dlgrno sequenes immeditely upstrem of TIS (n=736) or t internl CDS positions (n=8564) in the S. Typhimurium genome, ontriutions from eh motif window re sled to sum of one. Brhrts show the proportion of 5 or 3' rio-seq red ounts per nuleotide position, oloured y odon position. Hetmps show 5' or 3' rio-seq red ounts per length, oloured y z-sore per red length. () In reltion to SD motifs upstrem of initition odons. () In reltion to internl SD motifs within CDS regions. () In reltion to SD motifs upstrem of initition odons, feted y distne etween the SD motif nd initition odon (nt). 3

4 Supplementry_Fig_S3: Codon speifi red length distriutions. S. typhimurium rio-seq met-profiles in windows round the most ommonly used trnsltion initition odons t internl, in-frme CDS positions. Contriutions from eh odon window re sled to sum of one. (upper) Proportion of 5 or 3' rio-seq red ounts per nuleotide position, oloured y odon position. (lower) Hetmps of 5' or 3' rio-seq red ounts per frgment length, oloured y z-sore per red length. () ATG odons (n=35531). () GTG odons (n=35117). () TTG odons (n=17647). Supplementry_Fig_S4: Additionl predition support 4 () Showing the similrity in usge of different strt odons etween nnotted (n=4653) nd predited (n=4334) S. Typhimurium ORFs. (). Met plots showing the proportion of sled 5' rio-seq red ounts in reltion to nnotted or predited trnsltion initition sites, for ORFs mthing nnotted genes (n=3853), predited s extensions (n=214) or predited s truntions (n=205), in the S. Typhimurium dtset. Contriutions from eh gene re sled to sum of one. Nuleotide positions re oloured y odon position. Upstrem regions re highlighted in pink, downstrem regions re highlighted in light lue.

5 Supplementry_Fig_S5: Third odon periodiity nd GC ontent. () Proportion of 3' rio-seq red ounts per nuleotide position, from positions nt downstrem of the nnotted strt odon in the S. Typhimurium genome, oloured y odon position. Contriutions from eh gene re sled to sum of one. (upper) the highest 10% of regions y third odon GC ontent (n=467). (lower) the ottom 10% of regions y third odon GC ontent (n=468). () fourier trnsform showing the periodiity in the distriutions of (). Supplementry_Fig_S6: Evidene for predited novel trnsltion initition sites. () Sequene motifs reltion to predited trnsltion initition sites (n=61), for sequenes in ll novel predited ORFs in the S. Typhimurium dtset. (B) Met-profiles in reltion to nnotted (n=3853) or predited trnsltion initition sites. Blk dotted lines representing ORFs mthing nnotted genes, grey lines represent novel preditions. (upper) Met-profiles showing the perentge of GC ontent verged in 9nt sliding windows, higher vlues downstrem of the odon region re inditive of oding potentil. (lower) Met-profiles of free energy verged in 39nt sliding windows, higher vlues represent lower potentil for seondry struture formtion. 5

6 d e f Supplementry_Fig_S7: Rio-seq met profiles t strt odons - E. Coli Rio-seq met-profiles in windows round strt odons for nnotted genes (n=3726) in the E. oli genome, ontriutions from eh gene re sled to sum of one. (upper) Proportion of 5 or 3' rio-seq red ounts per nuleotide position, oloured y odon position. (lower) Hetmps of 5' or 3' rio-seq red length ounts, oloured y z-sore per red length. 6

7 Supplementry_Fig_S8: Lirry red length distriutions Red length distriutions of solute () or proportionl () ounts of ligned rio-seq reds per lirry. 7 Supplementry_Fig_S9: Red lengths djustments The sum of sled 5' rio-seq ounts from the () monosome or () polysome replite, in -30 to +60nt windows round nnotted strt odons (n=4205) in S. Typhimurium, per red length, oloured y odon position. Contriutions from eh gene re sled to sum of one. Blue rrows indite the pek orresponding to rio-seq footprints trnslting the strt odon.