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A direct comparison of the KB™ Basecaller and phred for identifying the bases from DNA sequencing using chain termination chemistry
BMC Research Notes volume 3, Article number: 257 (2010)
Abstract
Background
Relatively recently, the software KB™ Basecaller has replaced phred for identifying the bases from raw sequence data in DNA sequencing employing dideoxy chemistry. We have measured quantitatively the consequences of that change.
Results
The high quality sequence segment of reads derived from the KB™ Basecaller were, on average, 30-to-50 bases longer than reads derived from phred. However, microbe identification appeared to have been unaffected by the change in software.
Conclusions
We have demonstrated a modest, but statistically significant, superiority in high quality read length of the KB™ Basecaller compared to phred. We found no statistically significant difference between the numbers of microbial species identified from the sequence data.
Background
DNA sequencing by DNA polymerase chain termination was introduced by Sanger et al. [1] in 1977. In this technology, sequence is determined from the lengths of the terminated DNA chains. Electrophoresis is employed to separate the chains based upon length. A different fluorescent dye is covalently attached to each of the four dideoxy chain terminators. The presence of the dyes significantly affects the electrophoretic mobility of the chains. Therefore, sophisticated software must be employed to deconvolute the fluorescent signals into bases.
For some years, the suite of software of choice for DNA sequencing was introduced by Green and associates in 1998: phred for calling the bases in sequence reads, phrap for assembling the reads into contigs, and consed for displaying the contigs for editing [2–4]. Relatively recently, the manufacturer of the sequencing equipment, Applied Biosystems (ABI, Foster City, CA), introduced its own base calling software, the KB™ Basecaller, to replace phred http://www3.appliedbiosystems.com/cms/groups/mcb_marketing/documents/generaldocuments/cms_040412.pdf.
In our published study [5], we identified the microbes in the healthy adult human vagina by PCR amplifying the 16S ribosomal RNA genes, sequencing the genes with dideoxy chemistry, and identifying the microbes by comparison of the sequence to the data in the Ribosomal Database Project (RDP) [6]. We were concerned that the change in base-calling software would change the microbes identified. Primarily for this reason, we undertook a direct comparison of the KB™ Basecaller and phred, despite the fact that the comparison would be, and was, human labor intensive.
Results
Comparison of the high quality read lengths
The first comparison is of the high quality segments of the sequence reads derived from using phred to call the bases compared to the high quality segments of the sequence reads derived from using the KB™ Basecaller to call the bases. The results for all reads available for assembly are shown in Figure 1. We calculated the mean good quality read lengths for two cases: (1) sequence reads that were composed of a minimum of 100 consecutive high quality bases, because that is our minimum acceptable read length, and (2) reads that were composed of, at least, 500 good quality bases, because the longer the high quality segment, the more straightforward the assembly and the more secure the contig. These comparisons are shown in Table 1. For the first comparison (high quality read length > 100 bases), the KB™ Basecaller produced a mean read length of 763 bases with a standard deviation of 149 bases (n = 9,586), while phred produced a mean read length of 731 bases with a standard deviation of 116 bases (n = 9,572). Using the two sample t-test [7, 8], we concluded that the reads produced by the KB™ Basecaller were, on average, statistically significantly longer than the reads produced by phred (p-value < 10-15). For the second comparison (> 500 high quality bases), the KB™ Basecaller produced a mean read length of 804 bases with a standard deviation of 74 bases (n = 8,717), while phred produced a mean read length of 756 bases with a standard deviation of 71 bases (n = 8,911). Again, the reads produced by the KB™ Basecaller were, on average, statistically significantly longer than the reads produced by phred (p-value < 10-15). Thus, in agreement with a poster on the ABI website, on average, the KB™ Basecaller yields longer high quality segments than phred yields http://www3.appliedbiosystems.com/cms/groups/mcb_marketing/documents/generaldocuments/cms_040383.pdf. However, whereas the poster states that the KB™ Basecaller produces high quality segments an average of ~ 100 bases longer than phred, we find that the average difference is 30-to-50 bases. An unknown amount of this difference may be due to two different methods for determining the length of a high quality segment.
Microbe identification comparison
As an example of the sequence data processed to microbe species identification, the data for Project 95 are shown in Tables 2, 3, 4. Data for the other four projects are in the Additional files [Additional files 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12]. In all five projects, there are modest differences between the current microbe identifications and number of supporting reads compared to our published study [5]. We ascribe these differences to improvements in software and the data added to the RDP since 2005.
Six statistical comparisons were performed employing the Wilcoxon signed-rank test [9, 10]. The first comparison is of the total number of sequence reads in the five edited assemblies. The p-value is 0.1875. Thus, there is no statistically significant difference in the number of reads in the assemblies as produced by phred or the KB™ Basecaller. The second comparison is of the total number of microbial species identified. The p-value is 0.0625, which is the smallest p-value one can get using the Wilcoxon signed-rank test on five paired samples. While phred produced more species than the KB™ Basecaller in all five projects, this difference is not statistically significant.
Bacterial species from the genus Lactobacillus are the most common microbes found in the healthy adult vagina [e.g., [11]]. One of the five patients [Additional file 2, Supplemental Table S6] had (virtually) no Lactobacillus in her vagina, so the following comparison is among four projects. Consolidating all Lactobacillus species into the genus Lactobacillus, we compared the number of reads supporting the presence of Lactobacillus (p-value = 0.125). Thus, there is no statistically significant difference in the number of reads supporting the presence of Lactobacillus. A comparison of the number of reads supporting the presence of various Lactobacillus species (p-value = 0.125) and the number of different Lactobacillus species (p-value = 0.125) also failed to be statistically significant.
Conventionally, if the match of the sequence of the 16S ribosomal RNA gene to the closest sequence in the RDP is less than 97%, the microbial species is designated as novel [e.g., [12, 13]]. For our last comparison, we compared the number of reads supporting the presence of novel species and the number of novel species. Again, phred produced more reads and more novel species than the KB™ Basecaller in all five projects, but, as both p-values are 0.0625, there is no statistically significant difference.
Discussion
We have demonstrated a modest, but statistically significant, superiority in high quality read length of the KB™ Basecaller compared to phred. We found no statistically significant difference between the number of species identified from the sequence data processed starting with either software. Overall, it is gratifying that the two base-calling software led to the identification of the same microbes: i.e., microbe identification (our end product) is not a function of the base-calling software employed to call the bases from the raw sequence data.
Conclusions
We have demonstrated a modest, but statistically significant, superiority in high quality read length of the KB™ Basecaller compared to phred. We found no statistically significant difference between the numbers of microbial species identified from the sequence data.
Methods
We took the raw dideoxy sequence data from the last five women in our published study on vaginal microbes [5]. For the purposes of this comparison, we arbitrarily called them Projects 95-99. These sequences had already been deposited in the GenBank database [accession nos. AY958774-AY959212]. There are ~2,000 sequence reads for each woman. We started with the raw sequence data. In the first case, we called the bases with phred (incorporated in consed v14) [2]. A high quality base has a phred score (or equivalent) of 20 or higher [3]. In the second case, we called the bases with the KB™ Basecaller (v1.2; ABI, Foster City, California, USA). Standard default parameters were used for the two algorithms. It is possible that different results might have been achieved if different parameters had been employed. The accuracy of quality value assignment has not been examined. Any inaccuracies for either algorithm will have a direct impact on the average quality-trimmed read length. Thereafter, the sequence reads for both cases were processed in parallel with the same software. The sequence of each read was compared to the sequence of the plasmid vector. Plasmid bases were turned into "X"s. Because the plasmids had been grown in E. coli, the remaining sequence was compared to the sequence of E. coli DNA. By this process, a very few reads were removed from each dataset. Then, the reads were assembled into contigs by phrap [2, 3], and the contigs were displayed in consed [4]. Every contig in every one of the (now) ten projects was edited by hand and, for consistency, by the same person. There were three major types of manual edits. For (virtually) every recombinant plasmid, there was a forward read and a reverse read [5]. (1) Some contigs were composed of only forward (or reverse) reads. We call these "half contigs". For each read in a half contig, the opposing read was found and brought into the contig. (2) In some cases, the forward read was in one contig and the reverse read was in a different contig. The appropriate read of the pair was moved. (3) At a given position, some reads had high quality base "X" while other reads had high quality base "Y". The one contig was split into two contigs. Consed provides a consensus sequence for each contig [4]. For microbe identification, the contig consensus sequences were compared to the data in the RDP (release 10) [6].
Abbreviations
- N/A:
-
not applicable
- No.:
-
number
- RDP:
-
Ribosomal Database Project
- SD:
-
standard deviation.
References
Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Biotechnology. 1977, 24: 104-8.
Ewing B, Green P: Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 1998, 8: 186-94.
Ewing B, Hillier L, Wendl MC, Green P: Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 1998, 8: 175-85.
Gordon D, Abajian C, Green P: Consed: a graphical tool for sequence finishing. Genome Res. 1998, 8: 195-202.
Hyman RW, Fukushima M, Diamond L, Kumm J, Giudice LC, Davis RW: Microbes on the Human Vaginal Epithelium. Proc Natl Acad Sci USA. 2005, 102: 7952-7. 10.1073/pnas.0503236102.
Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM: The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res. 2009, 37: D141-D145. 10.1093/nar/gkn879.
Markowski CA, Markowski EP: Conditions for the Effectiveness of a Preliminary Test of Variance. The American Statistician. 1990, 44: 322-326. 10.2307/2684360.
David HA, Gunnink JL: The Paired t Test Under Artificial Pairing. The American Statistician. 1997, 51: 9-12. 10.2307/2684684.
Siegel S: Non-parametric statistics for the behavioral sciences. 1956, New York: McGraw-Hill, 75-83.
Wilcoxon F: Individual comparisons by ranking methods. Biometrics. 1945, 1: 80-83. 10.2307/3001968.
Nugent RP, Krohn MA, Hillier SL: Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol. 1991, 29: 297-301.
Stackebrandt E, Goebel BM: Taxonomic note: A place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol. 1994, 44: 846-849. 10.1099/00207713-44-4-846.
Gevers D, Cohan FM, Lawrence JG, Spratt BG, Coenye T, Feil EJ, Stackebrandt E, Van de Peer Y, Vandamme P, Thompson FL, Swings J: Opinion: Re-evaluating prokaryotic species. Nat Rev Microbiol. 2005, 3: 733-9. 10.1038/nrmicro1236.
Acknowledgements
This work was supported by National Human Genome Research Institute grant PO1 HG000205 (RWD).
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Authors' contributions
RWH conceived the comparison of the two base-calling software, hand edited all contigs, and wrote the manuscript. HJ performed the statistical evaluations of the data. MF employed the phred and ABI KB™ Basecaller software to call the bases, assembled the sequence reads with phrap, and displayed the contigs in consed. MF also compared the contig consensus sequences to the data in the RDP to identify the microbes and constructed the tables of microbes identified. RWD provided the intellectual, physical, and financial milieu for these experiments. All authors have read and approved the final manuscript.
Electronic supplementary material
13104_2010_661_MOESM1_ESM.XLS
Additional file 1: Table S5: Project 96: Microbes by genus. A table showing the microbes identified and the number (percent) of their supporting reads for Project 96. (XLS 16 KB)
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Additional file 2: Table S6: Project 96: Lactobacillus by species. A table showing the Lactobacillus species identified and the number (percent) of their supporting reads for Project 96. (XLS 14 KB)
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Additional file 3: Table S7: Project 96: Novel bacteria. A table showing the novel species identified, the closest named bacteria, and the number (percent) of their supporting reads for Project 96. (XLS 16 KB)
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Additional file 4: Table S8: Project 97: Microbes by genus. A table showing the microbes identified and the number (percent) of their supporting reads for Project 97. (XLS 16 KB)
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Additional file 5: Table S9: Project 97: Lactobacillus by species. A table showing the Lactobacillus species identified and the number (percent) of their supporting reads for Project 97. (XLS 14 KB)
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Additional file 6: Table S10: Project 97: Novel bacteria. A table showing the novel species identified, the closest named bacteria, and the number (percent) of their supporting reads for Project 97. (XLS 16 KB)
13104_2010_661_MOESM7_ESM.XLS
Additional file 7: Table S11: Project 98: Microbes by genus. A table showing the microbes identified and the number (percent) of their supporting reads for Project 98. (XLS 16 KB)
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Additional file 8: Table S12: Project 98: Lactobacillus by species. A table showing the Lactobacillus species identified and the number (percent) of their supporting reads for Project 98. (XLS 15 KB)
13104_2010_661_MOESM9_ESM.XLS
Additional file 9: Table S13: Project 98: Novel bacteria. A table showing the novel species identified, the closest named bacteria, and the number (percent) of their supporting reads for Project 98. (XLS 16 KB)
13104_2010_661_MOESM10_ESM.XLS
Additional file 10: Table S14: Project 99: Microbes by genus. A table showing the microbes identified and the number (percent) of their supporting reads for Project 99. (XLS 16 KB)
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Additional file 11: Table S15: Project 99: Lactobacillus by species. A table showing the Lactobacillus species identified and the number (percent) of their supporting reads for Project 99. (XLS 15 KB)
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Additional file 12: Table S16: Project 99: Novel bacteria. A table showing the novel species identified, the closest named bacteria, and the number (percent) of their supporting reads for Project 99. (XLS 15 KB)
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Hyman, R.W., Jiang, H., Fukushima, M. et al. A direct comparison of the KB™ Basecaller and phred for identifying the bases from DNA sequencing using chain termination chemistry. BMC Res Notes 3, 257 (2010). https://doi.org/10.1186/1756-0500-3-257
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DOI: https://doi.org/10.1186/1756-0500-3-257