illumina pyrosequencing

One aliquot was sequenced with the Roche 454 FLX Titanium sequencer (average read length, 450 bp) and the other one with the llumina GA II (100100 bp pair-ended reads) at Emory University Genomics Facility. 4, which is based on isolate genome data). Copyright: 2012 Luo et al. We found that homopolymer errors affected 2.132.78% and 0.321.02% of the total genes evaluated for the Lanier.454 and Lanier.Illumina data, respectively (dividing by the average gene length, 950 bp, provided the per base error rate; range was estimated from 100 replicates using Jackknife resampling), despite the fact that sequencing error in the raw reads of the two platforms was comparable (0.5% per base, in our hands). 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). We assessed homopolymer error rate in metagenomic data using two different strategies. Illumina-specific unique contig sequences (16 Mbp) were more than three times as many as the Roche 454-specific ones (5 Mbp), and these additional contigs were attributed to the larger Illumina dataset rather than sequencing artifacts or errors. 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). 2B, inset). PLOS ONE promises fair, rigorous peer review, We found a strong linear correlation (r2>0.99) between the Roche 454 and Illumina data with this respect (Fig. (C) Assemblies were obtained from 502 Mbp of Roche 454 and 2,460 Mbp of Illumina data using established protocols. (A) Length and coverage distribution of the contigs assembled from the Lanier.Illumina dataset. Specifically, in genomes of about 50% G+C content (similar to the 47% G+C of the Lake Lanier metagenome), Roche 454 assemblies showed about 5% more frameshift errors than those of Illumina assemblies. The DNA sample was divided into two aliquots of equal volume. LuoC, 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. The results presented here revealed the errors and limitations as well as the strengths in current metagenomics practice, and should constitute useful guidelines for experimental design and analysis. Contigs were defined as shared between the assemblies of the Lanier.454 and Lanier.Illumina data when they shared at least 95% nucleotide sequence identity and overlapped by at least 80% of their length (for the shorter contig). In order to account for possible biases introduced by uneven genus abundance and provide statistically robust estimates, we employed a Jackknifing resampling method. Analyzing raw (not assembled) reads, as opposed to assembled contigs, is typically restricted to cases where community complexity is too high or to specialized studies that aim to determine in situ abundance and/or population genetic structure and recombination [4], [10]. 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. 1). (A) Venn diagram showing the extent of overlapping and platform-specific raw reads between the Lanier.454 and Lanier.Illumina datasets (without assembly). 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). We performed six independent assemblies, using K=21, 25, 29 for the three SOAPdenovo runs and K=23, 27, 31 for the three Velvet runs. Lastly, our preliminary evaluation indicates that the latest Illumina sequencer (Hi-Seq 2000) performs similar to Illumina GA-II in terms of read length and quality; hence, our results should be applicable to this sequencer as well. As noted above, similar gap opening errors were observed for the metagenomic reads from the two platforms and single-base accuracy was comparable between the two platforms (99.34% vs. 99.46% for the Lanier.454 and Lanier.Illumina metagenomic reads, respectively). No, Is the Subject Area "Sequence alignment" applicable to this article? We would like to thank Chad Haase and Ryan Weil for their assistance with sequencing and Rachel Poretsky for critically reading the manuscript. The resulting datasets were 502 Mbp (Lanier.454) and 2,460 Mbp (Lanier.Illumina) in size; all our bioinformatic analyses and comparisons were based on these trimmed datasets. Assemblies of isolate genome sequences (closed or high-draft) were downloaded from the NCBI RefSeq database (called reference assemblies for convenience); raw Illumina and Roche 454 sequencing reads were available through the Joint Genome Institute (JGI, www.jgi.doe.gov). here. Our work also provides a methodology for evaluating and comparing metagenomic data from NGS platforms. Gene sequences from assembled contigs were extracted and ClustalW2 [31] was used to align the sequences against their orthologs from the reference assembly. No, Is the Subject Area "DNA sequencing" applicable to this article? We identified 0.4 million homopolymers (three identical consecutive nucleotide bases or more), of which 14 thousand (3.3% of the total) disagreed on length between the two assemblies, resulting in alternative amino acid sequences for about 7% of the total 72,709 gene sequences evaluated.

Most importantly, different tiles of the sequencing plate tend to produce reads of different quality [14], the 3 ends of sequences tend to have higher sequencing error rates compared to the 5 ends [15], and increased single-base errors have been observed in association with GGC motifs [16]. 1C); 57.7% and 49.5% of the total reads in the Lanier.Illumina and Lanier.454 datasets, respectively, were singletons (i.e., remained unassembled). Yes It is critical to assess the quality of the derived assemblies; to this end, several studies have recently attempted to evaluate the sequencing errors and artifacts specific to each NGS platform. It should be noted, however, that most of the previous error estimates and sequencing biases have been determined based on relatively simple DNA samples (e.g., a single viral genome) and thus, their relevance for complex community DNA samples remains to be evaluated. Performed the experiments: CL DT. In addition, given the monetary savings (e.g., we obtained the Illumina data for about one fourth of the cost of the Roche 454 data), Illumina, and short-read sequencing in general, may be a more appropriate method for metagenomic studies. Some of our results (e.g., assembly N50 comparisons, Fig. The higher sequence error rate observed for the TIGR reference genome might be due to the different strain of F. succinogenes sequenced or differences in the sequencing platforms or the assembly protocols used by JGI and TIGR. JS666 (-Proteobacteria), Polynucleobacter necessarius STIR1 (-Proteobacteria), Synechoccocus sp. We sampled 50% of the total homopolymers at random and estimated homolopolymer rate in this subset. First, we examined disagreements in gene sequences annotated on contigs larger than 500 bp and shared between the Lanier.454 and Lanier.Illumian assemblies. Consistent with the metagenomic observations, we found that Roche 454 assemblies from genome data contained a significantly higher portion of frameshift errors compared to Illumina assemblies from the same genome, when the assemblies were built with 5 times more Illumina data than the Roche 454 data, matching the relative ratio of the metagenomic data reported above. Finally, our evaluations showed that the choices of parameters and amount of input sequence of the assembly did not have any dramatic effect on the quality of the resulting contigs for both Illumina and Roche 454 assemblies (Fig. 1B). succinogenes S85, which was sequenced independently by The Institute for Genomic Research (TIGR GenBank accession: CP002158.1; JGI GenBank accession: CP001792.1). Consistent with these interpretations, we found that the single-base error of Illumina contigs increased by about 0.07% when we removed reads from the assembly so that the average coverage of the Illumina contigs would approximate the average coverage of the Roche 454 contigs (8). 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. https://doi.org/10.1371/journal.pone.0030087.g005, https://doi.org/10.1371/journal.pone.0030087.t001. Samples were collected from Lake Lanier, Atlanta, GA, below the Browns Bridge in August 2009 and community DNA was extracted as described previously [17]. Protein-coding genes encoded in the assembled contigs were identified by the MetaGene pipeline [26]. We aligned the assembled contigs from 9 Illumina and 8 Roche 454 assemblies from JGI data for the same genome against the TIGR reference assembly and calculated base call error rate and gap open error rate as described above for JGI genomes. More importantly, it is currently unclear how the above limitations affect the quality of the gene and genome sequences assembled from complex DNA samples, and whether the technologies provide different estimates of the genetic diversity in a sample due to their inherent chemistry and protocol differences. These findings call for special attention in cases where the sequenced DNA (e.g., community or isolate genome) is of low G+C%. For Lanier.Illumina, the SOAPdenovo [23] and Velvet [24] de novo assemblers were used to pre-assemble short reads into contigs using different K-mers. Yes For example, the high coverage of indigenous communities provided by NGS has made it possible to quantitatively assess the impact of diet on human gut microbiota [8] and the diversity of metabolic pathways within marine planktonic communities [9]. Graphs show the calculated base call error rate (A) and gap open error rate (B) for each comparison (figure key). The resulting contigs were merged into one dataset, and Newbler was used to assemble this dataset into longer contigs, using the same parameters as in the assembly of Lanier.454 data. correction. 4, 5, 6 and Table 1). Contributed reagents/materials/analysis tools: NK TR. We obtained (after trimming) a total of 502 Mbp (450 bp long reads) and 2,460 Mbp (100 bp pair-ended reads) from Roche 454 and Illumina sequencing, respectively, of the same community DNA sample. School of Biology and Center for Bioinformatics and Computational Genomics, Georgia Institute of Technology, Atlanta, Georgia, United States of America, PLOS ONE 7(3): 10.1371/annotation/64ba358f-a483-46c2-b224-eaa5b9a33939. 3), which is in agreement with previous results [5], [11]. We obtained a total of 513 Mbp and 3,640 Mbp Roche 454 and Illumina sequence data, respectively. 2). 4). 4), despite the fact that reads were trimmed based on the same quality standard prior to the analysis. Department of Human Genetics, Emory University, Atlanta, Georgia, United States of America, Affiliations Even though read lengths increase as the technologies advance, they are still far shorter than the desirable length (e.g., the average bacterial gene length is 950 bp) or the read length obtained from traditional Sanger sequencing (1000 bp). Our previous evaluation showed that our hybrid protocol outperforms other approaches for assembling metagenomic and genomic data [18]. We used the isolate genome data to evaluate the effect of the parameters of the assembly on the quality of the contigs as follows: a series of assemblies were obtained for genomes of low (Arcobacter nitrofigilis, 28%), medium (Fibrobacter succinogenes, 48%), and high (Cellulomonas flavigena, 74%) G+C% content. School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States of America, 29 Mar 2012: 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. We applied widely used protocols to assemble both sets of reads (see Materials and Methods for details), which substantially collapsed the Lanier.Illumina dataset into 57 Mbp of total unique sequences and the Lanier.454 dataset into 46 Mbp (Fig. For comparing gene calling accuracy on unassembled reads, we employed FragGeneScan [27] to predict genes on Lanier.454 and Lanier.Illumina reads using the 454 1% error rate model and the Illumina 0.5% error model, respectively. Base call errors and gap opening errors were identified as discrepancies between the read sequence and the reference assembly sequence using a custom Perl script. 2A, inset; and in [18]). More importantly, most of our findings from metagenomic data were reproducible in data from isolate genomes, which were sequenced by both sequencing platforms and showed a range of G+C% content (Figs. 2). These percentages were similar to those reported above based on the comparative method (the 3.3% of homopolymers that disagreed between the two datasets includes both Roche 454- and Illumina-specific homopolymer errors). Yes This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2B, inset) and this was primarily attributable to a higher sequencing error rate associated with A- and T-rich homopolymers (Fig. https://doi.org/10.1371/journal.pone.0030087.g001. here. To estimate the previously described errors associated with GGC motifs in Illumina reads [29], we selected the Roche 454 reads that were covered by at least 10 Illumina reads per base, on average, as reference sequences in Bowtie mapping (86.6 Mbp of reads in total). KyrpidesN, Competing interests: The authors have declared that no competing interests exist. Nine Illumina and eight Roche 454 assemblies from independent replicate datasets of the Fibrobacter succinogenes subsp. 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).

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