Abstract Introduction: Autism Spectrum Disorder (ASD) is a neurodevelopmental condition characterized by communication impairments, limited social interaction, restricted interests, repetitive behaviors and stereotypies. It manifests within the first 3 years of age and lasts for a lifetime with dramatic personal, familial and social consequences. ASD affects much more males than females (male:female=5:1) and its prevalence, that is continuously increasing, interests 2.24% of children and 1% of the general population [1,3]. Although genetics play a key role in ASD, its etiology is complex. Since most of individuals with ASD suffer from additional comorbidities including gastrointestinal disorders, intestinal permeability, inflammation and allergies, a gene-environment interaction has been proposed as ASD triggering [3]. Dysbiosis has been frequently associated with many neurological disorders including ASD. Thanks to the last advances in metagenomics, much progress has been made in the knowledge of gut microbiota profile and role, especially the prokaryotic one, but literature lacks of studies about eukaryotic colonizer of human intestine and their role in human health. Aims Here, employing different bioinformatics tools, we propose a metagenomics pilot study to define the prokaryotic and eukaryotic gut microbiota of children with ASD and neurotypical controls. The aims are to test different metagenomics pipelines and set up the more performing bioinformatics conditions to identify ASD microbial biomarkers useful for patient stratification and personalized treatments. Materials and Methods: We isolated DNA from stools collected from 6 children with ASD (5 males and 1 female) and 6 neurotypical controls matching for age and sex. Both 16S and 18S were amplified for each DNA and Illumina libraries prepared. NGS was performed by Illunima MiSeq platform coupled with Flowcell V3 2X300 and forward and reverse reading, reaching about 22Milion of sequences. As for bioinformatics analysis, three different software with several pipelines were applied: the automatic pipeline of SILVAngs analysis platform (https://ngs.arb-silva.de/silvangs/) [4], the MiSeq SOP pipeline of Mothur (https://mothur.org/) [5], and two pipelines of Qiime2 (https://qiime2.org/) [6]. The latter includes the Dada2 pipeline and the Deblur one. All the analyses were performed against SILVAv132 database [7], the only one that includes both 16s and 18S reference database. Results: The results obtained from the four tools are superimposable, also at different level of taxonomy detail. Restricting to Bacteria results, the identified genera are comparable with literature data [8]. As for Fungi, this first round of analysis doesn't return relevant differences between patients and controls. Further studies are needed to set up a pipeline specifically for the 18S datasets. Acknowledgements: EU project GEMMA (grant agreement No 825033), EPTRI and CNRBiOmics. Istituto San Vincenzo Erba and Albese, Italy
Microbiota Profile in Autism Spectrum Disorder: Different Metagenomics Approaches to analyze 16S and 18S rRNA
Chiappori F;Cupaioli FA;Mezzelani A
2020
Abstract
Abstract Introduction: Autism Spectrum Disorder (ASD) is a neurodevelopmental condition characterized by communication impairments, limited social interaction, restricted interests, repetitive behaviors and stereotypies. It manifests within the first 3 years of age and lasts for a lifetime with dramatic personal, familial and social consequences. ASD affects much more males than females (male:female=5:1) and its prevalence, that is continuously increasing, interests 2.24% of children and 1% of the general population [1,3]. Although genetics play a key role in ASD, its etiology is complex. Since most of individuals with ASD suffer from additional comorbidities including gastrointestinal disorders, intestinal permeability, inflammation and allergies, a gene-environment interaction has been proposed as ASD triggering [3]. Dysbiosis has been frequently associated with many neurological disorders including ASD. Thanks to the last advances in metagenomics, much progress has been made in the knowledge of gut microbiota profile and role, especially the prokaryotic one, but literature lacks of studies about eukaryotic colonizer of human intestine and their role in human health. Aims Here, employing different bioinformatics tools, we propose a metagenomics pilot study to define the prokaryotic and eukaryotic gut microbiota of children with ASD and neurotypical controls. The aims are to test different metagenomics pipelines and set up the more performing bioinformatics conditions to identify ASD microbial biomarkers useful for patient stratification and personalized treatments. Materials and Methods: We isolated DNA from stools collected from 6 children with ASD (5 males and 1 female) and 6 neurotypical controls matching for age and sex. Both 16S and 18S were amplified for each DNA and Illumina libraries prepared. NGS was performed by Illunima MiSeq platform coupled with Flowcell V3 2X300 and forward and reverse reading, reaching about 22Milion of sequences. As for bioinformatics analysis, three different software with several pipelines were applied: the automatic pipeline of SILVAngs analysis platform (https://ngs.arb-silva.de/silvangs/) [4], the MiSeq SOP pipeline of Mothur (https://mothur.org/) [5], and two pipelines of Qiime2 (https://qiime2.org/) [6]. The latter includes the Dada2 pipeline and the Deblur one. All the analyses were performed against SILVAv132 database [7], the only one that includes both 16s and 18S reference database. Results: The results obtained from the four tools are superimposable, also at different level of taxonomy detail. Restricting to Bacteria results, the identified genera are comparable with literature data [8]. As for Fungi, this first round of analysis doesn't return relevant differences between patients and controls. Further studies are needed to set up a pipeline specifically for the 18S datasets. Acknowledgements: EU project GEMMA (grant agreement No 825033), EPTRI and CNRBiOmics. Istituto San Vincenzo Erba and Albese, ItalyI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
