Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes

May combine two claims.

The apparent connection with RNA editing occurs also in insects; the RNA-edited potassium channel genes shaker, ether-a-go-go, and slowpoke each overlap four or more HCEs.

“The apparent connection with RNA editing occurs also in insects; the RNA-edited potassium channel genes shaker, ether-a-go-go, and slowpoke (Hoopengardner et al. 2003) each overlap four or more HCEs (see related observations by Glazov et al. 2005).”

• cites Hoopengardner et al. 2003 sourceNot checked — no full text of the source
• cites Glazov et al. 2005 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Earlier reports have described widespread conservation in 3' UTRs, and some have noted an enrichment for genes for DNA-binding proteins.

“These findings are consistent with earlier reports of widespread conservation in 3Ј UTRs (Duret et al. 1993;Lipman 1997), some of which have noted an enrichment for genes for DNA-binding proteins (Duret et al. 1993).”

• cites Duret et al. 1993 sourceSupported
• cites Lipman 1997 sourceSupported

May combine two claims.

Margulies et al. found that 72% of bases in predicted conserved elements in the CFTR region were not in exons, and Cooper et al. reported similar results based on whole-genome human/mouse/rat alignments.

“Margulies et al. (2003), using two different methods, found that 72% of bases in predicted conserved elements in the CFTR region were not in exons.Cooper et al. (2004) reported similar results based on wholegenome human/mouse/rat alignments.”

• cites Margulies et al. 2003Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Cooper et al. 2004 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Worm estimates of conserved fractions of coding, intronic, and intergenic regions are fairly similar to estimates from an early C. elegans/C. briggsae comparative study.

“In worms, our estimates of the fractions of coding regions, introns, and intergenic regions that are conserved are fairly similar to estimates based on an early comparative study of C. elegans and C. briggsae (Shabalina and Kondrashov 1999)”

• cites Shabalina and Kondrashov 1999 sourceSupported

May combine two claims.

The composition of vertebrate conserved elements agrees with bulk statistical estimates of the share of the human genome under selection and with an analysis of conserved elements in the CFTR region.

“These numbers are in good agreement both with bulk statistical estimates, based on genome-wide human/mouse and human/ mouse/rat alignments, of the share of the human genome that is under selection (Chiaromonte et al. 2003;Roskin et al. 2003;Cooper et al. 2004), and with an analysis of conserved elements in the region of the CFTR gene (Margulies et al. 2003;Thomas et al. 2003).”

• cites Chiaromonte et al. 2003 sourceNot checked — no full text of the source
• cites Roskin et al. 2003 sourceNot checked — no full text of the source
• cites Cooper et al. 2004 sourceSupported

May combine two claims.

MULTIZ shows good accuracy in simulation experiments, and synteny filters help ensure that only orthologous conserved elements are considered.

“This program shows good accuracy in simulation experiments (Blanchette et al. 2004), and our synteny filters help to ensure that only orthologous conserved elements are considered, but whole-genome alignment is a difficult problem, and programs that address it are still in their infancy.”

• cites Blanchette et al. 2004 sourceSupported

May combine two claims.

The remaining predicted elements cover 4.3% of the human genome, 44.5% of D. melanogaster, 26.4% of C. elegans, and 55.6% of S. cerevisiae.

“The remaining predicted elements cover 4.3% of the human genome, 44.5% of D. melanogaster, 26.4% of C. elegans, and 55.6% of S. cerevisiae.”

May combine two claims.

Different methods produced coverage estimates of 2.8%-8.1% for vertebrates, 36.9%-53.1% for insects, 18.4%-36.6% for worms, and 46.5%-67.6% for yeasts.

“Various different methods produced coverage estimates of 2.8%-8.1% for the vertebrates, 36.9%-53.1% for the insects, 18.4%-36.6%for the worms, and 46.5%-67.6%for the yeasts (see Supplemental material).”

May combine two claims.

The trend of extreme conservation in 3' UTRs is less pronounced in insects and was not observed in worms.

“This trend is less pronounced in insects and was not observed in worms.”

May combine two claims.

Introns of known genes contain 3.6% coverage by conserved bases, a smaller fraction than expected by chance.

“Introns of known genes and unannotated (putative intergenic) regions contain significant fractions of conserved bases (3.6% coverage for introns and 2.7% coverage for unannotated regions), but smaller fractions than would be expected by chance.”

May combine two claims.

The 1.18 million vertebrate elements cover 66% of bases in known coding regions, 23% of bases in known 5' UTRs, and 18% of bases in known 3' UTRs.

“The 1.18 million vertebrate elements, in addition to covering 66% of the bases in known coding regions (approximately the target level), cover 23% of the bases in known 5Ј UTRs and 18% of the bases in known 3Ј UTRs”

May combine two claims.

Predicted conserved elements cover roughly 3%-8% of the human genome, and substantially higher fractions of the more compact Drosophila melanogaster (37%-53%), Caenorhabditis elegans (18%-37%), and Saccharomyces cerevisiae (47%-68%) genomes.

“The predicted elements cover roughly 3%-8% of the human genome (depending on the details of the calibration procedure) and substantially higher fractions of the more compact Drosophila melanogaster (37%-53%), Caenorhabditis elegans (18%-37%), and Saccharaomyces cerevisiae (47%-68%) genomes.”

May combine two claims.

HCEs tend to be longer and less perfectly conserved than ultraconserved elements.

“These elements share certain properties with ultraconserved elements, but they tend to be longer and less perfectly conserved, and they overlap genes of somewhat different functional categories.”

May combine two claims.

Almost nine of ten (88%) known protein-coding exons are overlapped by predicted elements, as are 63% of 5'-UTR exons and 64% of 3'-UTR exons.

“Almost nine of 10 (88%) known protein-coding exons are overlapped by predicted elements, as well as almost two of three known UTR exons (63% of 5Ј-UTR exons and 64% of 3Ј-UTR exons”

May combine two claims.

About 1.31 million conserved elements were predicted for the vertebrate data set, about 472,000 for insects, about 98,000 for worms, and about 68,000 for yeasts.

“About 1.31 million conserved elements were predicted for the vertebrate data set, about 472,000 for the insects, about 98,000 for the worms, and about 68,000 for the yeasts.”

May combine two claims.

Unannotated putative intergenic regions contain 2.7% coverage by conserved bases, a smaller fraction than expected by chance.

“Introns of known genes and unannotated (putative intergenic) regions contain significant fractions of conserved bases (3.6% coverage for introns and 2.7% coverage for unannotated regions), but smaller fractions than would be expected by chance.”

May combine two claims.

Predicted elements include 42% of bases in a set of 561 putative RNA genes, and 56% of these genes are overlapped by predicted elements.

“The predicted elements also include 42% of the bases in a set of 561 putative RNA genes (see Methods), and 56% of these genes are overlapped by predicted elements”

May combine two claims.

In worms, UTRs and other mRNAs show substantially higher coverage by conserved elements than expected by chance, but this is not observed in insects.

“In all three cases, coding regions show substantially higher coverage than would be expected if conserved elements were distributed randomly, as do UTRs and other mRNAs in worms (but not in insects).”

May combine two claims.

If only RNA genes that align syntenically across species are considered, base-level coverage increases to 65%, about the same as in protein-coding genes.

“(If only RNA genes that align syntenically across species are considered, the base-level coverage increases to 65%, about the same as in protein-coding genes).”

May combine two claims.

In insects, worms, and yeasts, genes overlapped by HCEs in coding regions are enriched for some of the same GO categories as in vertebrates, but there are also substantial differences across species groups.

“In insects, worms, and yeasts, genes overlapped by HCEs in coding regions are enriched for some of the same GO categories as in vertebrates, but there are also substantial differences across species groups (Supplemental Table S4).”

May combine two claims.

HCEs identified for insects, worms, and yeasts cover 2.5%, 1.9%, and 8.0% of their respective reference genomes.

“The HCEs identified for the other three sets of genomes cover a higher percentage of each reference genome (2.5% in insect, 1.9% in worm, and 8.0% in yeast)”

May combine two claims.

HCE lengths range from 197 to 5783 bp (mean 627.9 bp) in insects, 622 to 12646 bp (mean 1889.6 bp) in worms, and 323 to 4005 bp (mean 973.5 bp) in yeasts.

“As with the vertebrates, the HCEs for the other three species groups are quite long, with lengths ranging from 197 to 5783 bp (mean 627.9 bp) for the insects, 622 to 12646 bp (mean 1889.6 bp) for the worms, and 323 to 4005 bp (mean 973.5 bp) for the yeasts.”

May combine two claims.

The RNA-editing site in GRIA2 corresponds to an HCE, as do the editing sites in related genes GRIA3 and GRIA4.

“Indeed, the RNA-editing site in GRIA2, shown in Figure 4, corresponds to an HCE, as do the editing sites in the related genes GRIA3 and GRIA4.”

May combine two claims.

Introns and unannotated regions show lower than expected coverage by conserved elements in all three compact species groups, but still appear to contain substantial numbers of conserved bases.

“Introns and unannotated regions show lower than expected coverage by conserved elements in all three species groups, but still appear to contain substantial numbers of conserved bases.”

May combine two claims.

The fractions of introns and intergenic regions in conserved elements are similar, with introns showing slightly higher fractions in all groups except yeast.

“The fractions of introns and intergenic regions in conserved elements are similar, with introns showing slightly higher fractions in all groups but yeast (where they are few in number).”

May combine two claims.

The fractions of HCEs in insects overlapping UTRs are similar to vertebrates (6.1% and 15.5% for 5' and 3' UTRs), but in worms these fractions are considerably lower (1.9% and 3.7%).

“The fractions of HCEs in insects overlapping UTRs are similar to those in vertebrates (6.1% and 15.5% overlap 5Ј and 3Ј UTRs, respectively), but in worm, these fractions are considerably lower (1.9% and 3.7%).”

May combine two claims.

The lengths of predicted elements for all four species groups are approximately geometrically distributed, averaging about 100-120 bp for vertebrates, insects, and yeasts, and about 270 bp for the less phylogenetically informative worm group.

“The lengths of the predicted elements for all four species groups are approximately geometrically distributed, averaging about 100-120 bp for the vertebrate, insect, and yeast groups and about 270 bp for the less phylogenetically informative worm group.”

May combine two claims.

Moving from vertebrates to insects to worms to yeasts, a progressively larger fraction of conserved elements falls in coding regions and UTRs, and a progressively smaller fraction falls in introns and unannotated regions.

“Moving from vertebrates to insects and then to worms and to yeasts, in decreasing order of genome size and general biological complexity, a progressively larger fraction of conserved elements can be seen to fall in coding regions and UTRs, and a progressively smaller fraction in introns and unannotated regions (Fig. 3).”

May combine two claims.

The fractions of elements in coding regions, UTRs, and other mRNAs tend to increase with score, while the fraction in introns tends to decrease.

“In particular, the fractions of elements in coding regions, UTRs, and other mRNAs tend to increase with score, while the fraction in introns tend to decrease.”

May combine two claims.

Only 42% of HCEs overlap known exons, with 36% overlapping CDS exons, 9% overlapping 5' UTR exons, and 16% overlapping 3' UTR exons; 19% fall completely in known introns and another 32% completely in unannotated regions.

“Nevertheless, only 42% of HCEs overlap known exons (36% overlap CDS exons, 9% overlap 5Ј UTR exons, and 16% overlap 3Ј UTR exons), with 19% falling completely in known introns, and another 32% completely in unannotated regions.”

May combine two claims.

In insects, 3'-UTR HCEs show statistically significant enrichment for secondary structure (P = 0.02), but 5'-UTR, intronic, and intergenic HCEs do not.

“Here, the 3Ј-UTR HCEs show a statistically significant enrichment for secondary structure (P = 0.02), but the 5Ј-UTR, intronic, and intergenic HCEs (for which the sample sizes are quite small) do not.”

May combine two claims.

In vertebrates, elements in ancestral repeats are shortest on average, those in introns and intergenic regions are slightly longer, those in UTRs are longer still, and those in CDSs are longest.

“A more detailed analysis in vertebrates revealed noticeable differences in the length distributions of the elements associated with different types of annotations; elements in ARs are shortest, on average, those in introns and intergenic regions are slightly longer, those in UTRs are longer still, and those in CDSs are longest (Supplemental Fig. S3).”

May combine two claims.

HCEs are like ultraconserved elements in showing extreme evolutionary conservation, but HCEs tend to be longer than UCEs and tend to have less extreme sequence conservation due to the length dependency of the log-odds scores.

“These highly conserved elements (HCEs) are like ultraconserved elements (UCEs) (Bejerano et al. 2004b) in that they show extreme evolutionary conservation (defined by some arbitrary threshold), but HCEs tend to be longer than UCEs and tend to have less extreme sequence conservation, due to the length dependency of the log-odds scores.”

• cites Bejerano et al. 2004bNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

At the base level, coding regions are enriched 22-fold for HCEs, while 3' UTRs and 5' UTRs are enriched 11-fold and eightfold, respectively.

“At the base level, coding regions are enriched 22-fold for HCEs, while 3Ј UTRs and 5Ј UTRs are enriched 11-fold and eightfold, respectively.”

May combine two claims.

In vertebrates, 18.0% of conserved bases fall in known coding regions, 1.1% in known 5' UTRs, and 3.6% in known 3' UTRs.

“In vertebrates, 18.0% of conserved bases fall in known coding regions (CDSs), 1.1% and 3.6% fall in known 5Ј and 3Ј UTRs, respectively”

May combine two claims.

In vertebrates, 5.2% of conserved bases fall in other mRNAs and 2.4% fall in other transcribed regions.

“and another 5.2% fall in other mRNAs.Another 2.4% fall in other transcribed regions, leaving about 70% unannotated.”

May combine two claims.

3' UTRs account for an unexpectedly large fraction of bases in vertebrate HCEs, and this trend becomes more pronounced as higher scoring subsets are considered: 9.6% in the top 5000, 12.5% in the top 1000, and 14.3% in the top 100, compared with 5.6% in all conserved elements.

“As noted above, 3Ј UTRs account for an unexpectedly large fraction of bases in vertebrate HCEs, and this trend becomes more pronounced as higher scoring subsets of all conserved elements are considered-3Ј UTRs account for 9.6% of bases in the top 5000 elements, 12.5% in the top 1000, and 14.3% in the top 100, compared with 5.6% in all conserved elements (Supplemental Fig. S3).”

May combine two claims.

Vertebrate HCEs are considerably longer on average than elements in the full set, with lengths ranging from 318 to 4922 bp and mean 781.4 bp.

“They are considerably longer on average than elements in the full set (lengths ranged from 318 to 4922 bp, with mean 781.4 bp)”

May combine two claims.

FPSs in intronic HCEs are on average about the same as those in 3'-UTR HCEs, while FPSs in intergenic HCEs are on average intermediate between those in 3'- and 5'-UTR HCEs.

“FPSs in intronic HCEs are, on average, about the same as those in 3Ј-UTR HCEs, while FPSs in intergenic HCEs are, on average, intermediate between those in 3Ј-and 5Ј-UTR HCEs.”

May combine two claims.

5' UTRs are only slightly overrepresented in HCEs (1.5% of bases, compared with 1.1% in all conserved elements), and they are almost absent in the top 100 elements.

“In contrast, 5Ј UTRs are only slightly overrepresented in HCEs (1.5% of bases, compared with 1.1% in all conserved elements), and they are almost absent in the top 100 elements.”

May combine two claims.

The set of vertebrate HCEs is about 10-fold larger than the set of UCEs and is based on a different set of species, including chicken and Fugu.

“In addition, the set of vertebrate HCEs is about 10-fold larger than the set of UCEs and is based on a different set of species (including chicken and Fugu).”

May combine two claims.

Most bases in ancestral repeats have evolved neutrally, and ancestral repeats are underrepresented fivefold in conserved elements.

“While most bases in ARs have evolved neutrally (ARs are underrepresented fivefold in conserved elements), some have apparently taken on critical functions that may help to differentiate mammals from ancestral vertebrates (Britten 1997;Jordan et al. 2003;van de Lagemaat et al. 2003).”

• AssumptionRests on an assumption: Most bases in mammalian ancestral repeats are neutrally evolving.

May combine two claims.

The multiple alignments consisted of blocks of local alignment covering 40.0% of the human genome, 86.9% of D. melanogaster, 43.8% of C. elegans, and 96.6% of S. cerevisiae.

“The multiple alignments consisted of blocks of local alignment covering 40.0% of the human genome, 86.9% of D. melanogaster, 43.8% of C. elegans, and 96.6% of S. cerevisiae (Supplemental Table S2).”

May combine two claims.

Even if HCEs are excluded when computing CNF pc, phastCons high-CNF segments overlap 13% of all HCEs and 18% of intronic/intergenic HCEs, enrichments of eightfold and 13-fold, respectively.

“Even if redefined such that the HCEs are excluded when computing the CNF pc , the phastCons high-CNF segments overlap 13% of all HCEs and 18% of intronic/intergenic HCEsenrichments of eightfold and 13-fold, respectively.”

May combine two claims.

A preliminary analysis of a larger set of insect genomes found that total coverage by conserved elements and the fraction of conserved elements in noncoding regions both decreased somewhat, but did not change dramatically; general properties of conserved elements and HCEs were essentially unchanged.

“We have carried out a preliminary analysis of a larger set of insect genomes, including draft assemblies of D. ananassae, D. virilis, and D. mojavensis, and found that the total coverage by conserved elements and the fraction of conserved elements in noncoding regions both de-creased somewhat, but did not change dramatically; the general properties of conserved elements and HCEs were essentially”

• AssumptionThe authors state this as an assumption: Coding regions evolve in fundamentally similar ways across species groups, more similar than noncoding regions.
• AssumptionThe authors state this as an assumption: The fraction of sites in coding regions that are conserved is not highly sensitive to the phylogeny.

May combine two claims.

These independently defined phastCons high-CNF segments also include 23% of human/rodent ultraconserved elements, a 15-fold enrichment.

“These independently defined phastCons high-CNF segments also include 23% of human/rodent ultraconserved elements, a 15-fold enrichment.”

May combine two claims.

In the human GRIA2 gene, peaks in the conservation plot generally correspond to exons and valleys to noncoding regions, but a 158-bp conserved noncoding element can be seen near the 3' end of exon 11.

“Peaks in the conservation plot generally correspond to exons and valleys to noncoding regions, but a 158-bp conserved noncoding element can be seen near the 3Ј end of exon 11.”

May combine two claims.

The fractions of conserved bases that fall in protein-coding regions are lowest for vertebrates (11%-24%), slightly higher for insects (26%-27%), substantially higher for worms (49%-60%), and highest for yeasts (84%-87%).

“In addition, the fractions of conserved bases that fall in protein-coding regions are lowest for vertebrates (11%-24%), slightly higher for insects (26%-27%), substantially higher for worms (49%-60%), and highest for yeasts (84%-87%).”

• AssumptionThe authors state this as an assumption: Coding regions evolve in fundamentally similar ways across species groups, more similar than noncoding regions.
• AssumptionThe authors state this as an assumption: The fraction of sites in coding regions that are conserved is not highly sensitive to the phylogeny.

May combine two claims.

Unlike human/chicken high-CNF segments, phastCons high-CNF segments are not significantly depleted for genes, but like human/chicken high-CNF segments, they show a significant enrichment for trans-dev genes.

“Unlike human/ chicken high-CNF segments, these segments are not significantly depleted for genes, but like human/chicken high-CNF segments, they show a significant enrichment for trans-dev genes.”

May combine two claims.

A fourth GAL4-binding site is reflected by a small bump in the conservation scores, as is the promoter itself.

“A fourth GAL4-binding site also is reflected by a small bump in the conservation scores (left arrow), as is the promoter itself (right arrow).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The 451 Rfam seed sequences mapped perfectly to 561 positions in the human genome.

“(The 451 sequences mapped perfectly to 561 positions in the genome.)”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In the base-level display of GRIA2, several synonymous substitutions are visible on the left and the elevated conservation abruptly ends after the splice site, while on the right there are fewer synonymous substitutions and the elevated conservation extends into the intron.

“On the left, several synonymous substitutions are visible (highlighted bases) and the elevated conservation abruptly ends after the splice site, while on the right, there are fewer synonymous substitutions and the elevated conservation extends into the intron.”

May combine two claims.

In all three comparisons of FPS distributions (HCEs in 3' UTRs vs random 3' UTRs, HCEs in 3' UTRs vs HCEs in 5' UTRs, and HCEs in introns vs HCEs in coding regions), the difference between the distributions is highly statistically significant (P = 8.8e-66, P = 1.1e-8, and P = 4.4e-215, respectively; Wilcoxon rank sum test).

“In all three cases, the difference between the distributions is highly statistically significant (P = 8.8 e-66, P = 1.1 e-8, and P = 4.4 e-215, respectively; Wilcoxon rank sum test).”

May combine two claims.

Stable gene deserts account for only 12% of bases in intergenic regions, yet 53% of the 1578 intergenic HCEs fall within or overlap stable deserts, 4.5 times the expected number.

“Stable gene deserts account for only 12% of bases in intergenic regions, yet 53% of the 1578 intergenic HCEs fall within or overlap stable deserts, 4.5 times the expected number.”

May combine two claims.

There are 101 phastCons high-CNF segments covering 2.1% of the human genome and averaging 601 kb in length and 13.8% CNF pc.

“There are 101 phastCons high-CNF segments covering 2.1% of the human genome and averaging 601 kb in length and 13.8% CNF pc .”

May combine two claims.

75% of stable deserts include or are overlapped by at least one HCE, while this is true for only 15% of variable deserts.

“Conversely, 75% of stable deserts include or are overlapped by at least one HCE, while this is true for only 15% of variable deserts.”

May combine two claims.

The fractions of bases in each reference genome conserved across related species are smallest for vertebrates (3%-8%), intermediate for worms and insects (18%-37% and 37%-53%, respectively), and largest for yeasts (47%-68%).

“In particular, the fractions of bases in each reference genome that are conserved across related species are smallest for vertebrates (3%-8%), intermediate for worms and insects (18%-37% and 37%-53%, respectively), and largest for yeasts (47%-68%).”

• AssumptionThe authors state this as an assumption: Coding regions evolve in fundamentally similar ways across species groups, more similar than noncoding regions.
• AssumptionThe authors state this as an assumption: The fraction of sites in coding regions that are conserved is not highly sensitive to the phylogeny.

May combine two claims.

Strong cross-species conservation can be seen in the regulatory region upstream of the promoter, as well as in the protein-coding portion of the S. cerevisiae GAL1 gene.

“Strong cross-species conservation can be seen in the regulatory region upstream of the promoter, as well as in the protein-coding portion of the gene.”

May combine two claims.

Variable gene deserts account for 30% of bases in intergenic regions and only 2.2% of intergenic HCEs fall within or overlap variable deserts.

“In contrast, variable gene deserts account for 30% of bases in intergenic regions and only 2.2% of intergenic HCEs fall within or overlap variable deserts.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Most groups have used pairwise alignments and simple, percent identity-based methods for identifying conserved elements.

“Most groups have used pairwise alignments and simple, percent identity-based methods for identifying conserved elements.”

Classified as probably not a substantial claim.

Most methods, including phylogenetic shadowing, use a sliding window of fixed size, which can be a limitation.

“In addition, most methods (including phylogenetic shadowing) use a sliding window of fixed size, which can be a limitation.”

May combine two claims.

Only the phylogenetic shadowing method makes use of branch lengths of the phylogeny, allows multiple substitutions per site on single branches, and considers the pattern of substitution.

“only the “phylogenetic shadowing” method (Boffelli et al. 2003) (to our knowledge) makes use of the branch lengths of the phylogeny, allows for multiple substitutions per site on single branches of the tree, and considers the “pattern” of substitution”

• cites Boffelli et al. 2003 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

The results are generally not highly sensitive to the precise level of target coverage used in the calibration procedure.

“Our results are, in general, not highly sensitive to the precise level of target coverage used in this calibration procedure (see Supplemental material).”

Classified as probably not a substantial claim.

Model parameters were constrained so that coverage of known coding regions by predicted conserved elements was equivalent in all species groups.

“we constrained the model parameters such that the coverage of known coding regions by predicted conserved elements (i.e., the fraction of coding bases falling in conserved elements) was equivalent in all groups.”

May combine two claims.

The method does not depend on fourfold degenerate sites being free from selection or being suitable proxies for neutrally evolving sites, and as an unsupervised learning method it is not dependent on possibly incomplete or erroneous annotations.

“it does not depend on 4d sites being free from selection or being suitable proxies for neutrally evolving sites in general; and as an “unsupervised” learning method (see Methods), it is not dependent on possibly incomplete and/or erroneous annotations.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The worm results may be influenced by difficulty aligning noncoding regions in C. elegans and C. briggsae and by limited phylogenetic information in pairwise alignments.

“Note that the results for worm may be influenced by the difficulty of aligning noncoding regions in C. elegans and C. briggsae and by the limited phylogenetic information in pairwise alignments (see Discussion and Supplemental material).”

May combine two claims.

The trend underscores the importance of noncoding regions in the genomes of complex eukaryotes, whose complexity apparently derives not so much from increased numbers of protein-coding genes as from more elaborate mechanisms for gene regulation.

“but it nevertheless underscores the importance of noncoding regions in the genomes of complex eukaryotes, whose complexity apparently derives not so much from increased numbers of proteincoding genes as from more elaborate mechanisms for gene regulation.”

May combine two claims.

SCFGs are richer computational models than HMMs and can accommodate long-distance base pairing in RNA structures.

“SCFGs are richer computational models than HMMs, which can accommodate the long distance base pairing that occurs in RNA structures (Durbin et al. 1998).”

• cites Durbin et al. 1998Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing).

Correlations between HCEs and predicted miRNA target sites must be treated cautiously because they may be artifacts of considering conservation in target-site prediction.

“Correlations between HCEs and predicted miRNA target sites must be treated cautiously, because they may be artifacts of considering conservation in target-site prediction.”

May combine two claims.

Three groups of known RNA-binding proteins and the mRNAs they bind provide circumstantial evidence for a connection between HCEs in 3' UTRs and post-transcriptional regulation, and, if predictions of target sites are accurate, for a connection with miRNAs.

“Three groups of known RNA-binding proteins and the mRNAs they bind provide further circumstantial evidence for a connection between HCEs in 3Ј UTRs and post-transcriptional regulation, and moreover (if predictions of target sites are accurate), for a connection with miRNAs.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The fraction of conserved elements in introns and intergenic regions may be underestimated for the two-species worm data set.

“Note that the fraction of conserved elements in introns and intergenic regions may be underestimated for the twospecies worm data set (see Discussion and Supplemental material).”

Classified as probably not a substantial claim.

The FPS is designed to avoid biases related to base composition, overall conservation level, and sequence length.

“The FPS reflects possible (local) stem-pairings within each sequence in a multiple alignment and compensatory substitutions along the branches of the phylogeny, but is designed to avoid biases related to base composition, overall conservation level, and sequence length (see Methods and Supplemental material).”

May combine two claims.

The fraction of HCEs overlapping known exons is somewhat higher than the 23% observed for UCEs, presumably because of the length dependency of the log-odds scores and the tolerance for a small number of substitutions.

“The fraction of HCEs overlapping known exons is somewhat higher than the 23% observed for UCEs (Bejerano et al. 2004b), presumably because of the length depen-dency of the log-odds scores and the tolerance for a small number of substitutions.”

• cites Bejerano et al. 2004bNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

MiR-196 is unusual among known animal miRNAs for its near-perfect complementarity to its HOXB8 target site and for inducing cleavage of the HOXB8 mRNA rather than inhibiting translation.

“MiR-196 is unusual among known animal miRNAs for its near-perfect complementarity to its HOXB8 target site and for inducing cleavage of the HOXB8 mRNA rather than inhibiting translation (Yekta et al. 2004).”

• cites Yekta et al. 2004 sourceNot checked — no full text of the source

May combine two claims.

Having only two species reduced phylogenetic information per site, forcing the tuning parameter (expected length) to be increased, causing short conserved elements to be missed and larger numbers of nonconserved bases to be contained within predicted conserved elements.

“In addition, having only two species considerably reduced the amount of phylogenetic information per site, forcing the tuning parameter (expected length) to be increased, and in turn, causing short, conserved elements to tend to be missed, and larger numbers of nonconserved bases to be contained within predicted conserved elements (see Methods).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

No correction for multiple testing was performed, but because there were fewer than 1000 individual tests, P < 5e-5 can be considered significant.

“No correction for multiple testing was performed, but there were fewer than 1000 individual tests, so P < 5 e-5 can be considered significant.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Using different window sizes, relaxing the constraints on columns with missing data, and using the Kolmogorov-Smirnov test instead of the Wilcoxon test did not produce significant differences in the results.

“Using different window sizes, relaxing the constraints on columns with missing data and using the Kolmogorov-Smirnov test instead of the Wilcoxon test did not produce significant differences in our results.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Similar evidence was found for insect HCEs.

“Similar evidence was found for insect HCEs.”

Probably not a substantial claim: may not pass S4 (based).

More complex parameterizations increase the danger of converging on biologically uninteresting local maxima of the likelihood function.

“More complex parameterizations also increase the danger of converging on biologically uninteresting local maxima of the likelihood function.”

• AssumptionThe authors state this as an assumption: All sites evolve at one of two evolutionary rates: conserved and nonconserved.
• AssumptionThe authors state this as an assumption: The two evolutionary rates are uniform across the genome.
• AssumptionThe authors state this as an assumption: Sites evolve independently conditional on whether they are in conserved or nonconserved regions.
• AssumptionThe authors state this as an assumption: The phylogenetic models for conserved and nonconserved regions have the same branch-length proportions, base compositions, and substitution patterns.

Probably not a substantial claim: may not pass S3 (load-bearing).

Misplacements of alignment gaps may cause regions to appear more or less conserved than they really are.

“Furthermore, misplacements of alignment gaps may cause regions to appear more or less conserved than they really are.”

May combine two claims.

Statistical enrichments and individual cases suggest that at least some vertebrate HCEs function as cis-regulatory binding sites, as RNA genes, in mRNA secondary structures important for RNA editing or post-transcriptional regulation, or as microRNA targets.

“As with ultraconserved elements, the reasons for the extreme conservation observed in most vertebrate HCEs remain unknown, but statistical enrichments and individual cases suggest that at least some of these sequences function as cisregulatory binding sites, as RNA genes, in mRNA secondary structures important for RNA editing or post-transcriptional regulation, or as microRNA targets.”

Classified as probably not a substantial claim.

The entire analysis is conditional on whole-genome alignments produced by the MULTIZ program.

“Finally, it is important to note that our entire analysis is conditional on whole-genome alignments produced by the MULTIZ program.”

May combine two claims.

Some conserved elements are undoubtedly missed because of alignment failures, and even with synteny filtering, other predicted elements are probably spurious in that they are based on alignments of nonorthologous sequence.

“Some conserved elements are undoubtedly missed because of alignment failures, and even with synteny filtering, other predicted elements are probably spurious in that they are based on alignments of nonorthologous sequence.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Some false matches are undoubtedly included in the set of putative RNA genes identified from Rfam seed sequences.

“Only exact matches were retained, but many of these genes are short and some tend to match repeats, so some false matches are undoubtedly included in this set.”

May combine two claims.

The individual parameter estimates were fairly consistent and their averages should be close to true genome-wide m.l.e.'s.

“The individual parameter estimates were fairly consistent and their averages should be close to true genome-wide m.l.e.'s (see Supplemental material).”

Classified as probably not a substantial claim.

The smoothness constraint, requiring the phylogenetic information threshold (PIT) to be the same for all species groups, was designed to ensure that predicted conserved elements were supported by similar minimal amounts of phylogenetic evidence, taking into consideration differences in phylogenetic information per site.

“This constraint was designed to ensure that predicted conserved elements were supported by similar (minimal) amounts of phylogenetic evidence, taking into consideration differences in phylogenetic information per site.”

Classified as probably not a substantial claim.

Possible biases related to alignment gaps and missing data were avoided by constraining the structures such that alignment columns with >50% gaps or missing data had to fall in nonpairing regions.

“Possible biases related to alignment gaps and missing data were avoided by constraining the structures such that alignment columns with >50% gaps or missing data had to fall in nonpairing regions.”

May combine two claims.

These conserved elements tend to be short compared with those in 3' UTRs, and may fail to be identified as HCEs simply for this reason.

“These elements tend to be short, e.g., compared with those in 3Ј UTRs, and may fail to be identified as HCEs simply for this reason.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The graphs in Figure 3 change somewhat, but not dramatically, under alternative calibration methods.

“Note that these graphs change somewhat (but not dramatically) under alternative calibration methods (see Supplemental material).”

Classified as probably not a substantial claim. May combine two claims.

The phylogenetic models in sp and nsp for stem-pairing and nonpairing positions were trained on known RNA structures from Rfam, but were forced to have equal expected rates of substitution to avoid biases related to conservation level.

“The phylogenetic models in sp and nsp for stem-pairing and nonpairing positions were trained on known RNA structures from Rfam (Griffiths-Jones et al. 2003), but were forced to have equal expected rates of substitution to avoid biases related to conservation level.”

• cites Griffiths-Jones et al. 2003Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

These factors have probably resulted in an overestimate of the fraction of conserved bases that fall in coding regions (estimated at 49%-60%), and may have resulted in an underestimate of the total fraction conserved (estimated at 18%-37%).

“Together, these factors have probably resulted in an overestimate of the fraction of conserved bases that fall in coding regions (estimated at 49%-60%), and may have resulted in an underestimate of the total fraction conserved (estimated at 18%-37%).”

• AssumptionThe authors state this as an assumption: Coding regions evolve in fundamentally similar ways across species groups, more similar than noncoding regions.
• AssumptionThe authors state this as an assumption: The fraction of sites in coding regions that are conserved is not highly sensitive to the phylogeny.

May combine two claims.

HCEs are substantially enriched in stable gene deserts and depleted in variable gene deserts, and most stable deserts have HCEs, while most variable deserts do not.

“Thus, HCEs are substantially enriched in stable gene deserts and depleted in variable gene deserts, and most stable deserts have HCEs, while most variable deserts do not.”

May combine two claims.

Fragments with sparse data (<1000 sites aligned) were excluded, but the amount of data discarded was negligible, and the average parameter estimates can reasonably be said to represent the entire genome-wide data sets.

“Fragments with sparse data (<1000 sites aligned) were excluded from this procedure, but the amount of data discarded in this way was negligible, and the average parameter estimates can reasonably be said to represent the entire genome-wide data sets.”

May combine two claims.

The paper's results generally support previous estimates of the fraction of the human genome under selection and the fraction of conserved human bases that fall in noncoding regions, and allow approximate quantitative comparison across species groups.

“Our results generally support previous estimates of the fraction of the human genome under selection and the fraction of conserved human bases that fall in noncoding regions, and they allow for an approximate quantitative comparison of these fractions across species groups.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

For practical reasons, exact maximum likelihood estimates of free parameters were not obtained for entire genome-wide data sets; instead, genome-wide alignments were divided into fragments of at most about 1 Mb and parameters estimated separately for each fragment, then averaged.

“For practical reasons, exact maximum likelihood estimates (m.l.e.'s) of free parameters were not obtained for entire genomewide data sets, but instead, the genome-wide alignments were divided into fragments of, at most, about 1 Mb in length (in the coordinate system of the reference genome), and parameters were estimated separately for each fragment.”

Classified as probably not a substantial claim.

To avoid length biases, the folding potential score (FPS) was computed locally in a sliding window of 150 bp (step size 50 bp) rather than scoring whole UTRs.

“To avoid length biases, we did not score whole UTRs, but instead, computed the FPS locally in a sliding window of 150 bp (step size 50 bp).”

Classified as probably not a substantial claim. May combine two claims.

The rate matrix in nsp is a marginalized version of the rate matrix in sp, so that the two models make use of the same nucleotide distribution.

“In addition, the rate matrix in nsp is a marginalized version of the rate matrix in sp , so that the two models make use of the same nucleotide distribution.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Treating alignment gaps as missing data ignores an important source of phylogenetic information.

“In addition, treating alignment gaps as missing data ignores an important source of phylogenetic information.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The phylo-HMM used by phastCons is a fairly rich probabilistic model but is clearly not realistic in several respects.

“The phylo-HMM used by phastCons is a fairly rich probabilistic model, but it is clearly not realistic in several respects.”

• AssumptionThe authors state this as an assumption: All sites evolve at one of two evolutionary rates: conserved and nonconserved.
• AssumptionThe authors state this as an assumption: The two evolutionary rates are uniform across the genome.
• AssumptionThe authors state this as an assumption: Sites evolve independently conditional on whether they are in conserved or nonconserved regions.
• AssumptionThe authors state this as an assumption: The phylogenetic models for conserved and nonconserved regions have the same branch-length proportions, base compositions, and substitution patterns.

May combine two claims.

The large degree of divergence between C. elegans and C. briggsae led to low-alignment coverage and may have created a bias toward alignment of conserved elements in coding rather than noncoding regions.

“The large degree of divergence between C. elegans and C. briggsae led to low-alignment coverage, and may have created a bias toward alignment of conserved elements in coding rather than noncoding regions (because conserved noncoding regions tend to be shorter on average; hence, harder to align.)”

May combine two claims.

Alternative calibration methods, based on full maximum-likelihood parameter estimation, estimation of neutral rates from fourfold degenerate sites, and alternative coverage targets in coding regions, have led to generally similar results, and certain basic conclusions appear fairly robust.

“Nevertheless, alternative calibration methods-based on full maximumlikelihood parameter estimation, estimation of neutral rates from fourfold degenerate sites, and alternative coverage targets in coding regions-have led to generally similar results (see Supplemental material), and certain basic conclusions appear to be fairly robust.”

• AssumptionThe authors state this as an assumption: Coding regions evolve in fundamentally similar ways across species groups, more similar than noncoding regions.
• AssumptionThe authors state this as an assumption: The fraction of sites in coding regions that are conserved is not highly sensitive to the phylogeny.