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AmpliconDuo: A Split-Sample Filtering Protocol for High-Throughput Amplicon Sequencing of Microbial Communities.

Lange A, Jost S, Heider D, Bock C, Budeus B, Schilling E, Strittmatter A, Boenigk J, Hoffmann D - PLoS ONE (2015)

Bottom Line: Further, we discard sequences that are not found in both branches ("AmpliconDuo filter").The filter does not distort overall apparent community compositions.Finally, we quantitatively explain the effect of the AmpliconDuo filter by a simple mathematical model.

View Article: PubMed Central - PubMed

Affiliation: Research Group Bioinformatics, Faculty of Biology, University of Duisburg-Essen, Essen, Germany.

ABSTRACT
High throughput sequencing (HTSeq) of small ribosomal subunit amplicons has the potential for a comprehensive characterization of microbial community compositions, down to rare species. However, the error-prone nature of the multi-step experimental process requires that the resulting raw sequences are subjected to quality control procedures. These procedures often involve an abundance cutoff for rare sequences or clustering of sequences, both of which limit genetic resolution. Here we propose a simple experimental protocol that retains the high genetic resolution granted by HTSeq methods while effectively removing many low abundance sequences that are likely due to PCR and sequencing errors. According to this protocol, we split samples and submit both halves to independent PCR and sequencing runs. The resulting sequence data is graphically and quantitatively characterized by the discordance between the two experimental branches, allowing for a quick identification of problematic samples. Further, we discard sequences that are not found in both branches ("AmpliconDuo filter"). We show that the majority of sequences removed in this way, mostly low abundance but also some higher abundance sequences, show features expected from random modifications of true sequences as introduced by PCR and sequencing errors. On the other hand, the filter retains many low abundance sequences observed in both branches and thus provides a more reliable census of the rare biosphere. We find that the AmpliconDuo filter increases biological resolution as it increases apparent community similarity between biologically similar communities, while it does not affect apparent community similarities between biologically dissimilar communities. The filter does not distort overall apparent community compositions. Finally, we quantitatively explain the effect of the AmpliconDuo filter by a simple mathematical model.

No MeSH data available.


Related in: MedlinePlus

Effect of AmpliconDuo filtering on chimeras for prokaryotic sample Pro2.Chimeras defined by being recognized by UCHIME in de novo mode with score ≥ 1. Top: Frequency of chimeras in branches A, B of split sample as function of their read numbers, before (dashed lines) and after (solid lines) application of AmpliconDuo filter. Bottom: Fraction of chimeras passing the AmpliconDuo filter (ffiltered/funfiltered) for read numbers 1 to 20 in both branches A, B, and corresponding prediction P(riA, riB ≥ 1) using the Poisson model in Eq (8) with λi = 1, 2, …, 20.
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pone.0141590.g008: Effect of AmpliconDuo filtering on chimeras for prokaryotic sample Pro2.Chimeras defined by being recognized by UCHIME in de novo mode with score ≥ 1. Top: Frequency of chimeras in branches A, B of split sample as function of their read numbers, before (dashed lines) and after (solid lines) application of AmpliconDuo filter. Bottom: Fraction of chimeras passing the AmpliconDuo filter (ffiltered/funfiltered) for read numbers 1 to 20 in both branches A, B, and corresponding prediction P(riA, riB ≥ 1) using the Poisson model in Eq (8) with λi = 1, 2, …, 20.

Mentions: When we studied the effect of the AmpliconDuo filter on the prokaryotic chimeras, we found that the AmpliconDuo filter removed only low abundance chimeras, and even there the removal was incomplete. Generally, effectiveness of the AmpliconDuo filter for chimera removal decreased with increasing chimera abundance and was negligible for sequences with read numbers of ten or more. The top part of Fig 8 illustrates this for a prokaryotic sample (see S5 Fig for a detailed breakdown of samples Pro1 and Pro3).


AmpliconDuo: A Split-Sample Filtering Protocol for High-Throughput Amplicon Sequencing of Microbial Communities.

Lange A, Jost S, Heider D, Bock C, Budeus B, Schilling E, Strittmatter A, Boenigk J, Hoffmann D - PLoS ONE (2015)

Effect of AmpliconDuo filtering on chimeras for prokaryotic sample Pro2.Chimeras defined by being recognized by UCHIME in de novo mode with score ≥ 1. Top: Frequency of chimeras in branches A, B of split sample as function of their read numbers, before (dashed lines) and after (solid lines) application of AmpliconDuo filter. Bottom: Fraction of chimeras passing the AmpliconDuo filter (ffiltered/funfiltered) for read numbers 1 to 20 in both branches A, B, and corresponding prediction P(riA, riB ≥ 1) using the Poisson model in Eq (8) with λi = 1, 2, …, 20.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4629888&req=5

pone.0141590.g008: Effect of AmpliconDuo filtering on chimeras for prokaryotic sample Pro2.Chimeras defined by being recognized by UCHIME in de novo mode with score ≥ 1. Top: Frequency of chimeras in branches A, B of split sample as function of their read numbers, before (dashed lines) and after (solid lines) application of AmpliconDuo filter. Bottom: Fraction of chimeras passing the AmpliconDuo filter (ffiltered/funfiltered) for read numbers 1 to 20 in both branches A, B, and corresponding prediction P(riA, riB ≥ 1) using the Poisson model in Eq (8) with λi = 1, 2, …, 20.
Mentions: When we studied the effect of the AmpliconDuo filter on the prokaryotic chimeras, we found that the AmpliconDuo filter removed only low abundance chimeras, and even there the removal was incomplete. Generally, effectiveness of the AmpliconDuo filter for chimera removal decreased with increasing chimera abundance and was negligible for sequences with read numbers of ten or more. The top part of Fig 8 illustrates this for a prokaryotic sample (see S5 Fig for a detailed breakdown of samples Pro1 and Pro3).

Bottom Line: Further, we discard sequences that are not found in both branches ("AmpliconDuo filter").The filter does not distort overall apparent community compositions.Finally, we quantitatively explain the effect of the AmpliconDuo filter by a simple mathematical model.

View Article: PubMed Central - PubMed

Affiliation: Research Group Bioinformatics, Faculty of Biology, University of Duisburg-Essen, Essen, Germany.

ABSTRACT
High throughput sequencing (HTSeq) of small ribosomal subunit amplicons has the potential for a comprehensive characterization of microbial community compositions, down to rare species. However, the error-prone nature of the multi-step experimental process requires that the resulting raw sequences are subjected to quality control procedures. These procedures often involve an abundance cutoff for rare sequences or clustering of sequences, both of which limit genetic resolution. Here we propose a simple experimental protocol that retains the high genetic resolution granted by HTSeq methods while effectively removing many low abundance sequences that are likely due to PCR and sequencing errors. According to this protocol, we split samples and submit both halves to independent PCR and sequencing runs. The resulting sequence data is graphically and quantitatively characterized by the discordance between the two experimental branches, allowing for a quick identification of problematic samples. Further, we discard sequences that are not found in both branches ("AmpliconDuo filter"). We show that the majority of sequences removed in this way, mostly low abundance but also some higher abundance sequences, show features expected from random modifications of true sequences as introduced by PCR and sequencing errors. On the other hand, the filter retains many low abundance sequences observed in both branches and thus provides a more reliable census of the rare biosphere. We find that the AmpliconDuo filter increases biological resolution as it increases apparent community similarity between biologically similar communities, while it does not affect apparent community similarities between biologically dissimilar communities. The filter does not distort overall apparent community compositions. Finally, we quantitatively explain the effect of the AmpliconDuo filter by a simple mathematical model.

No MeSH data available.


Related in: MedlinePlus