title: Groundtruthing next-gen sequencing for microbial ecology-biases and errors in community structure estimates from PCR amplicon pyrosequencing

authors: Lee, Charles Kai-Wu

subjects: RNA 16S

abstract: Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa-a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3-V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with >1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure.

text: Groundtruthing next-gen sequencing for microbial ecology-biases and errors in community structure estimates from PCR amplicon pyrosequencing Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa-a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3-V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with >1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure.

year: 2012

title: Exploring the ‘how’ and ‘why’ of value orientations in Physical Education teacher education

authors: Gillespie, Lorna B.

subjects: Physical Education teacher education

abstract: This paper focuses on the significance of curriculum value orientations for curriculum implementation and, therefore, for teacher education. The paper draws on data arising from research undertaken with six Health and Physical Education teachers in Aotearoa New Zealand to explore issues pertinent to Physical Education teacher education (PETE). Selected findings from the study are presented and critically engaged with from a teacher education viewpoint to specifically address (i) considerations relating to students’ value orientations that teacher educators need to be cognisant of, and (ii) issues arising for teacher educators seeking to engage with value orientations in undergraduate and/or post-graduate PETE programmes. The paper foregrounds the highly personal nature of value orientations in considering their significance in teacher education programmes and is written from the position of the author as a teacher educator committed to raising student teachers’ awareness of value orientations and their influence on curriculum and pedagogy. Attention is drawn to the significance of course content, practicum, critically reflective practice, and programme structures in relation to efforts to embed value orientations into teacher education programmes.

text: Exploring the ‘how’ and ‘why’ of value orientations in Physical Education teacher education This paper focuses on the significance of curriculum value orientations for curriculum implementation and, therefore, for teacher education. The paper draws on data arising from research undertaken with six Health and Physical Education teachers in Aotearoa New Zealand to explore issues pertinent to Physical Education teacher education (PETE). Selected findings from the study are presented and critically engaged with from a teacher education viewpoint to specifically address (i) considerations relating to students’ value orientations that teacher educators need to be cognisant of, and (ii) issues arising for teacher educators seeking to engage with value orientations in undergraduate and/or post-graduate PETE programmes. The paper foregrounds the highly personal nature of value orientations in considering their significance in teacher education programmes and is written from the position of the author as a teacher educator committed to raising student teachers’ awareness of value orientations and their influence on curriculum and pedagogy. Attention is drawn to the significance of course content, practicum, critically reflective practice, and programme structures in relation to efforts to embed value orientations into teacher education programmes.

year: 2011

title: Rangatiratanga - Kawanatanga: Dealing with rhetoric

authors: Nikora, Linda Waimarie

abstract: Numbered among the few Maori appointed to an academic position during the late 1980s, I have seen our discipline and profession, in all its awkward self-consciousness, make genuine attempts to grapple the respond to questions and challenges delivered by Maori and Pakeha alike.  For example, Where is the Maori content in courses?  Why aren’t Maori graduating from professional training programmes?  Why are there not more Maori academics? Where is the place/space for Maori as practitioners, academics, researchers and decision-makers?  Why does it seem that Maori are always on the ‘client’ side of the professional/client equation?  Where are the Maori men?  In responding to these persistent challenges the professional and discipline in Aotearoa/New Zealand has been irreversibly changed, in my opinion.  The change has not been dramatic (psychology is somewhat averse to self-promotion) but, of those changes that have occurred, a definite contribution has been made to advancing Maori development, to building a more bicultural discipline and practice, and to supporting the strength of the Kawanatanga and Rangatiratanga dynamic whose importance I emphasize here.

text: Rangatiratanga - Kawanatanga: Dealing with rhetoric Numbered among the few Maori appointed to an academic position during the late 1980s, I have seen our discipline and profession, in all its awkward self-consciousness, make genuine attempts to grapple the respond to questions and challenges delivered by Maori and Pakeha alike.  For example, Where is the Maori content in courses?  Why aren’t Maori graduating from professional training programmes?  Why are there not more Maori academics? Where is the place/space for Maori as practitioners, academics, researchers and decision-makers?  Why does it seem that Maori are always on the ‘client’ side of the professional/client equation?  Where are the Maori men?  In responding to these persistent challenges the professional and discipline in Aotearoa/New Zealand has been irreversibly changed, in my opinion.  The change has not been dramatic (psychology is somewhat averse to self-promotion) but, of those changes that have occurred, a definite contribution has been made to advancing Maori development, to building a more bicultural discipline and practice, and to supporting the strength of the Kawanatanga and Rangatiratanga dynamic whose importance I emphasize here.

year: 2001