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Direct Comparisons of Illumina vs. Roche 454 Sequencing Technologies on the Same Microbial Community DNA Sample

Luo, Chengwei, Tsementzi, Despina, Kyrpides, Nikos, Read, Timothy, Konstantinidis, Konstantinos T.·2012·PLoS ONE
DOI 10.1371/journal.pone.0030087PMC PMC3277595
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Figure 1 Genetic diversity and gene abundance in Roche 454 vs. Illumina data. (A) Venn diagram showing the extent of overlapping and platform-specific raw reads between the Lanier.454 and Lanier.Illumina datasets (without assembly). (B) Protein sequences annotated on raw (not assembled) reads matched genes in the reference assembly more frequently for the Roche 454 than the Illumina data. Conversely, protein sequences annotated on Illumina reads more frequently matched to the wrong protein sequence in the reference assembly (mismatched genes) or did not match any reference gene (unmatched genes). (C) Assemblies were obtained from 502 Mbp of Roche 454 and 2,460 Mbp of Illumina data using established protocols. Venn diagram showing the extent of overlapping and platform-specific sequences of assembled contigs longer than 500 bp. (D) Number of Roche 454 (x-axis) and Illumina (y-axis) reads mapping on the same contig shared between the two assemblies.
Figure 1 Genetic diversity and gene abundance in Roche 454 vs. Illumina data. (A) Venn diagram showing the extent of overlapping and platform-specific raw reads between the Lanier.454 and Lanier.Illumina datasets (without assembly). (B) Protein sequences annotated on raw (not assembled) reads matched genes in the reference assembly more frequently for the Roche 454 than the Illumina data. Conversely, protein sequences annotated on Illumina reads more frequently matched to the wrong protein sequence in the reference assembly (mismatched genes) or did not match any reference gene (unmatched genes). (C) Assemblies were obtained from 502 Mbp of Roche 454 and 2,460 Mbp of Illumina data using established protocols. Venn diagram showing the extent of overlapping and platform-specific sequences of assembled contigs longer than 500 bp. (D) Number of Roche 454 (x-axis) and Illumina (y-axis) reads mapping on the same contig shared between the two assemblies.CC BY · PubMed Central
Figure 2 Average length and sequence accuracy comparisons of the Roche 454 and Illumina assembled contigs. (A) Length and coverage distribution of the contigs assembled from the Lanier.Illumina dataset. Note that contigs shorter than 500 bp (red) were numerically more abundant than longer contigs (green) but were characterized by substantially lower coverage (inset). (B) Graph shows the comparison of the contig length of three assemblies plotted against the N statistic of the assembly [for instance, N40 (x-axis) is equal to about 1 Kbp (y-axis), which means that (100−40 = 60) % of the entire assembly is contained in contigs no shorter than 1 Kbp]. Due to frameshifts caused primarily by homopolymer-associated errors in the derived consensus sequence of the contigs, genes from Roche 454 assembly had fewer complete matches in the NR database relatively to their Illumina counterparts (inset; results are based on a total of 72,709 gene sequences annotated on contigs that were shared between the two assemblies and were longer than 500 bp).
Figure 2 Average length and sequence accuracy comparisons of the Roche 454 and Illumina assembled contigs. (A) Length and coverage distribution of the contigs assembled from the Lanier.Illumina dataset. Note that contigs shorter than 500 bp (red) were numerically more abundant than longer contigs (green) but were characterized by substantially lower coverage (inset). (B) Graph shows the comparison of the contig length of three assemblies plotted against the N statistic of the assembly [for instance, N40 (x-axis) is equal to about 1 Kbp (y-axis), which means that (100−40 = 60) % of the entire assembly is contained in contigs no shorter than 1 Kbp]. Due to frameshifts caused primarily by homopolymer-associated errors in the derived consensus sequence of the contigs, genes from Roche 454 assembly had fewer complete matches in the NR database relatively to their Illumina counterparts (inset; results are based on a total of 72,709 gene sequences annotated on contigs that were shared between the two assemblies and were longer than 500 bp).CC BY · PubMed Central
Figure 3 Characteristics of homopolymer-related sequence errors in Roche 454 metagenome assembly. (A) A's and T's contribute significantly more homopolymer errors than C's and G's. The average G+C% content of the metagenome was 47.4%; thus, our results are not simply attributable to higher abundance of A's and T's in the metagenome. (B) Error rate (as a percentage of the total genes evaluated, y-axis) increases as homopolymer length increases (x-axis).
Figure 3 Characteristics of homopolymer-related sequence errors in Roche 454 metagenome assembly. (A) A's and T's contribute significantly more homopolymer errors than C's and G's. The average G+C% content of the metagenome was 47.4%; thus, our results are not simply attributable to higher abundance of A's and T's in the metagenome. (B) Error rate (as a percentage of the total genes evaluated, y-axis) increases as homopolymer length increases (x-axis).CC BY · PubMed Central
Figure 4 Roche 454 and Illumina GA II read sequence quality based on isolate genome data. Roche 454 sequencing quality is evaluated in panels A through D, which show: (A) base call error rate of individual reads (x-axis) for each genome evaluated (y-axis); (B) base call error rate (y-axis) plotted against the G+C% of the genome; (C) gap opening error rate of individual reads (x-axis) for each genome evaluated (y-axis); (D) gap opening error rate (y-axis) plotted against the G+C% of the genome. Illumina GA II sequencing quality is evaluated in panels E and F, which show: (E) base call error rate of individual reads plotted against the G+C% of the genome; and (F) gap opening error rate of individual reads plotted against the G+C% of the genome. Panels A and C represent the variation observed in reads from different (replicate) datasets of the same genome; red bars represent the median, the upper and lower box boundaries represent the upper and lower quartiles, and the upper and lower whiskers represent the largest and smallest observations. All 2D plots (panels B, D, E, and F) represent the arithmetic average of the medians of each dataset for the same genome; Illumina medians were identical among replicate datasets; therefore, only one value is shown in panel E. The results show that Illumina sequence quality was affected less than that of Roche 454 by the G+C% content of the sequenced DNA (note the lower r-squared value and the slope in E). Thus, the results reported for Illumina based on the metagenome of Lake Lanier (47 G+C%) should be also applicable to metagenomes with different G+C% contents.
Figure 4 Roche 454 and Illumina GA II read sequence quality based on isolate genome data. Roche 454 sequencing quality is evaluated in panels A through D, which show: (A) base call error rate of individual reads (x-axis) for each genome evaluated (y-axis); (B) base call error rate (y-axis) plotted against the G+C% of the genome; (C) gap opening error rate of individual reads (x-axis) for each genome evaluated (y-axis); (D) gap opening error rate (y-axis) plotted against the G+C% of the genome. Illumina GA II sequencing quality is evaluated in panels E and F, which show: (E) base call error rate of individual reads plotted against the G+C% of the genome; and (F) gap opening error rate of individual reads plotted against the G+C% of the genome. Panels A and C represent the variation observed in reads from different (replicate) datasets of the same genome; red bars represent the median, the upper and lower box boundaries represent the upper and lower quartiles, and the upper and lower whiskers represent the largest and smallest observations. All 2D plots (panels B, D, E, and F) represent the arithmetic average of the medians of each dataset for the same genome; Illumina medians were identical among replicate datasets; therefore, only one value is shown in panel E. The results show that Illumina sequence quality was affected less than that of Roche 454 by the G+C% content of the sequenced DNA (note the lower r-squared value and the slope in E). Thus, the results reported for Illumina based on the metagenome of Lake Lanier (47 G+C%) should be also applicable to metagenomes with different G+C% contents.CC BY · PubMed Central
Figure 5 Percentage of reference genome recovered by Illumina (yellow) and Roche 454 (green) assemblies. Graph shows the variation observed in assemblies from different (replicate) datasets of the same genome; red bars represent the median, the upper and lower box boundaries represent the upper and lower quartiles, and the upper and lower whiskers represent the largest and smallest observations. Note that Illumina assemblies recovered a significantly larger fraction of the reference genome than Roche 454 assemblies (two tailed Whitney-Mann U test p-value = 0.014), which is consistent with the results from the metagenomes (Fig. 2). The results for the isolate genomes were based on Illumina input reads that were about 5 times as many as the Roche 454 input reads to provide a ratio that was similar to that of the metagenomic comparisons (5∶1).
Figure 5 Percentage of reference genome recovered by Illumina (yellow) and Roche 454 (green) assemblies. Graph shows the variation observed in assemblies from different (replicate) datasets of the same genome; red bars represent the median, the upper and lower box boundaries represent the upper and lower quartiles, and the upper and lower whiskers represent the largest and smallest observations. Note that Illumina assemblies recovered a significantly larger fraction of the reference genome than Roche 454 assemblies (two tailed Whitney-Mann U test p-value = 0.014), which is consistent with the results from the metagenomes (Fig. 2). The results for the isolate genomes were based on Illumina input reads that were about 5 times as many as the Roche 454 input reads to provide a ratio that was similar to that of the metagenomic comparisons (5∶1).CC BY · PubMed Central
Figure 6 Comparisons of Illumina and Roche 454 assemblies against an independently sequenced reference genome. Nine Illumina and eight Roche 454 assemblies from independent replicate datasets of the Fibrobacter succinogenes subsp. succinogenes S85 genome sequenced at JGI were compared against the reference assemblies from the JGI and TIGR genome projects of Fibrobacter succinogenes subsp. succinogenes S85. Graphs show the calculated base call error rate (A) and gap open error rate (B) for each comparison (figure key). Red bars represent the median, the upper and lower box boundaries represent the upper and lower quartiles, and the upper and lower whiskers represent the largest and smallest observations.
Figure 6 Comparisons of Illumina and Roche 454 assemblies against an independently sequenced reference genome. Nine Illumina and eight Roche 454 assemblies from independent replicate datasets of the Fibrobacter succinogenes subsp. succinogenes S85 genome sequenced at JGI were compared against the reference assemblies from the JGI and TIGR genome projects of Fibrobacter succinogenes subsp. succinogenes S85. Graphs show the calculated base call error rate (A) and gap open error rate (B) for each comparison (figure key). Red bars represent the median, the upper and lower box boundaries represent the upper and lower quartiles, and the upper and lower whiskers represent the largest and smallest observations.CC BY · PubMed Central
Figure 7 Dependence of the quality of assembled contigs on the parameters of the Illumina assembly. Assembly parameters (primary and secondary x-axes) were evaluated for low (Arcobacter nitrofigilis, 28%; left), medium (Fibrobacter succinogenes, 48%; middle), and high (Cellulomonas flavigena, 74%; right) G+C% genomes. For each genome, a 2D-grid assembly was performed, varying the size of input sequences (20×, 30×, 40×, …, 130×) and the K-mer (21, 23, 25, …, 37) of each of the assemblers used (SOAPdenovo and Velvet). The quality of the resulting contigs was examined in terms of base call error (A) and gap opening error (B), which revealed that the combination of the parameters of the assembly did not have a dramatic effect on the quality of the contigs (see projected contours on x-z and y-z space). Similarly for the Roche 454 data, a 2D-grid assembly was performed, varying the size of input sequences (20×, 30×, 40×, …, 130×) and the minimal aligned length to merge contigs or reads (30 bp, 40 bp, …, 100 bp) for Newbler. The quality of the resulting contigs was examined in terms of base call error (C) and gap opening error (D), which revealed that the combination of the parameters of the assembly did not have a dramatic effect on the quality of the contigs except in the extreme values of the minimal aligned length (see projected contours on x-z and y-z space), which were avoided in our direct comparisons of Illumina versus Roche 454 assemblies.
Figure 7 Dependence of the quality of assembled contigs on the parameters of the Illumina assembly. Assembly parameters (primary and secondary x-axes) were evaluated for low (Arcobacter nitrofigilis, 28%; left), medium (Fibrobacter succinogenes, 48%; middle), and high (Cellulomonas flavigena, 74%; right) G+C% genomes. For each genome, a 2D-grid assembly was performed, varying the size of input sequences (20×, 30×, 40×, …, 130×) and the K-mer (21, 23, 25, …, 37) of each of the assemblers used (SOAPdenovo and Velvet). The quality of the resulting contigs was examined in terms of base call error (A) and gap opening error (B), which revealed that the combination of the parameters of the assembly did not have a dramatic effect on the quality of the contigs (see projected contours on x-z and y-z space). Similarly for the Roche 454 data, a 2D-grid assembly was performed, varying the size of input sequences (20×, 30×, 40×, …, 130×) and the minimal aligned length to merge contigs or reads (30 bp, 40 bp, …, 100 bp) for Newbler. The quality of the resulting contigs was examined in terms of base call error (C) and gap opening error (D), which revealed that the combination of the parameters of the assembly did not have a dramatic effect on the quality of the contigs except in the extreme values of the minimal aligned length (see projected contours on x-z and y-z space), which were avoided in our direct comparisons of Illumina versus Roche 454 assemblies.CC BY · PubMed Central

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Research works printed but not linked: 25

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Metadata

Authors
Luo, C., Tsementzi, D., Kyrpides, N., Read, T., & Konstantinidis, K. T.
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2012-02-10
Volume
7
Issue
2
Pages
e30087
ISSN
1932-6203
DOI
10.1371/journal.pone.0030087
PMC
PMC3277595
Cited by
337
References
6
Language
en
Update notices
correction (2012-03-29)
Licence
creativecommons.org/licenses/by/4.0/ (unspecified)

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