697 697
697
Joanna Bybee1, Rojelio Mejia2,Ruben Cimino3, Ashish Damania2, Rebecca Jen3, Patricia Bryan3, Alejandro Krolewiek3, Barton E. Slatko1 1New England Biolabs Inc., Ipswich MA USA 2Na)onal School of Tropical Medicine, Baylor college of Medicine, Houston TX USA 3Universidad Nacional de Salta, Salta Province, Argen)na
Abstract
Next genera3on sequencing (NGS) for microbiome analysis is commonly performed using 16S rRNA gene sequencing or whole genome shotgun sequencing (WGS). We carried out both WGS and 16S sequencing on human fecal samples from a 122 Argen3nian cohort study focusing on two groups: helminth infected (Ascaris, Ancylostoma, Necator, Strongyloides, and Trichuris) versus non-‐infected (no-‐parasite) individuals verified by mul3-‐parallel real-‐3me quan3ta3ve PCR. WGS approach provided higher resolu3on allowing classifica3on to the bacterial strain level and in some cases even sub-‐strain level. 16S sequencing could not provide resolu3on below genus level. Both methods demonstrated similar sensi3vity to detect Shannon alpha diversity differences. While there were no sta3s3cal differences within the helminth infected group (p = 0.999) or no-‐parasite group (p = 0.400), WGS showed a significant increase in difference of means (DOM) as compared to 16S rRNA gene sequencing. DOM provides a measure of the change in propor3on of specific bacterial sequences for helminth and no-‐parasite groups. This measure is useful for determining the capacity of an assay to discriminate between 2 experimental groups and small effect size. The WGS method provides rich metagenomic func3onal informa3on as compared to 16S rRNA sequencing. Metagenomic func3onal informa3on for 16S rRNA reads can be inferred using PICRUST so`ware through taxonomic informa3on, but it lacks the direct evidence of genes found in WGS. On the other hand, 16S sequencing is computa3onally inexpensive, while WGS data are challenging to manage/analyse and require so`ware with complex algorithms. Our results show that WGS offers higher taxonomic resolu3on and discrimina3on along with metagenomic func3onal informa3on while 16S provides a reasonable op3on if the taxonomic informa3on is the primary focus of a study. This study provides important informa3on for selec3ng the op3mal assay based on func3on and price with implica3ons in evolu3onary inves3ga3ons and tropical medicine.
• Asymptoma3c children • Ages 4-‐6 years old • No recent an3bio3cs • qPCR and microscopy for presence of : Ascaris lumbricoides Strongyloides stercoralis Ancylostoma duodenale Giardia lamblia Necator americanus Cryptosporidium species Trichuris trichiura Entamoeba histoly;ca • NEBNext® Microbiome DNA Enrichment Kit • NEBNext® Ultra™ DNA Library Prep Kit for Illumina® • Illumina NextSeq® Whole genome sequencing • Livermore Metagenomics Analysis Toolkit (LMAT) and Diamond so`ware • Phred quality score 20 (99% base call accuracy) • Normalized to 10,000 reads for bacterial diversity
0 0.01 0.1
Proportion of sequences (%)
Proportion of sequences (%)
1
Giardia Non-Giardia
1
10
100 1000 10000
Giardia fg/µl • Greater than 1 fg/µl Giardia DNA has significant decrease in bacterial diversity to No Parasite group (p = 0.0244) 5
Giardia Helminths No Parasites Giardia Helminth
• Giardia infec3ons had lower cellular amino acid metabolic processes than helminth infec3ons (p = 0.047)
4
3
• f 2
1
• Higher Giardia burdens correlate to less bacterial diversity which could indicate worse disease status
• Giardia infected group had significant increases in Prevotella species compared to helminth groups • Coinfec3ons negated those differences • Metagenomics showed lower cellular amino acid processes and decreased cobalamin (Vitamin B12) biosynthesis genes in Giardia infected children and related to high Giardia burden
• Useful for epidemiology and morbidity studies 0 o Giardia > 1 fg/µl No Giardia Helminths • Correlate mechanism of l Giardia< 1 fg/µl Parasites Helminth decreased Vitamin B12 genes to growth delays in • High Giardia infected children • Giardia >1 fg/µl group had more children infected with had decreased cobalamin abundant Prevotella than No Parasite intes3nal parasites biosynthesis genes compared to group p = 0.037 (A) with Helminths No Parasites (p = 0.038)(A) with • Expanding understanding group decreased Prevotella to Giardia of morbidity and compensatory effects from group (p = 0.024) and Giardia/helminth malnutri3on Helminth infec3ons (p = co-‐infected nega3ng these differences 0.021)(B) B A (p = 0 .019) ( B) B • Future direc3ons: A • Correlate quan3ty of parasite DNA with clinical outcomes • Associate morbidity to changes in microbiome • Treat children with an3-‐ parasi3cs and evaluate changes in microbiome Propor3on of sequences (%)
• 99 pa3ent samples
2
Conclusions
• These findings are mostly with higher Giardia burdens and likely due to changes in intes3nal micro-‐environments due to Giardia crea3ng anaerobic microenvironments
Propor3on of sequences (%)
• Temperate climate
3
Propor3on oof f sequences sequences (%) (%) Propor3on
• Peri-‐urban community
Spearman r = -0.5491 p = 0.0244
Prevotella propor3on (%)
• Field site: Orán, Argen3na
4
Shannon Alpha Diversity
Materials and methods
• Giardia group had more anaerobic bacteria than other cohorts op3mizing condi3ons for Prevotella (p = 0.012)
• Increasing Giardia burden (fg/µl) correlates to decreasing intes3nal bacterial diversity
Prevotella propor3on (%)
• >2 billion GI parasite infec3ons worldwide in poorest and resource-‐ deprived communi3es • GI parasites may disrupt normal intes3nal microbiota • Decreased microbial biodiversity is associated with disease, including: § Malabsorp3on § Inflammatory bowel diseases • Vitamin B12 involved in metabolism of every human cell • Bacteria have the enzymes needed for vitamin B12 biosynthesis • qPCR is rapid, quan3ta3ve, high-‐ throughput and is a more reliable species-‐specific method
Results
Shannon Alpha Diversity
Introduc)on
Acknowledgements
Funding for this project was provided by the Na3onal School of Tropical Medicine, Baylor College of Medicine and New England Biolabs, Inc.
High Giardia Low Giardia No Parasites
High Giardia Low Giardia No Parasites
Giardia Helminths No Parasites Giardia High Giardia Low Giardia No Parasites Giardia Helminths No Giardia Parasites Helminth Helminth
697
Joanna Bybee1, Rojelio Mejia2,Ruben Cimino3, Ashish Damania2, Rebecca Jen3, Patricia Bryan3, Alejandro Krolewiek3, Barton E. Slatko1 1New England Biolabs Inc., Ipswich MA USA 2Na)onal School of Tropical Medicine, Baylor college of Medicine, Houston TX USA 3Universidad Nacional de Salta, Salta Province, Argen)na
Abstract
Next genera3on sequencing (NGS) for microbiome analysis is commonly performed using 16S rRNA gene sequencing or whole genome shotgun sequencing (WGS). We carried out both WGS and 16S sequencing on human fecal samples from a 122 Argen3nian cohort study focusing on two groups: helminth infected (Ascaris, Ancylostoma, Necator, Strongyloides, and Trichuris) versus non-‐infected (no-‐parasite) individuals verified by mul3-‐parallel real-‐3me quan3ta3ve PCR. WGS approach provided higher resolu3on allowing classifica3on to the bacterial strain level and in some cases even sub-‐strain level. 16S sequencing could not provide resolu3on below genus level. Both methods demonstrated similar sensi3vity to detect Shannon alpha diversity differences. While there were no sta3s3cal differences within the helminth infected group (p = 0.999) or no-‐parasite group (p = 0.400), WGS showed a significant increase in difference of means (DOM) as compared to 16S rRNA gene sequencing. DOM provides a measure of the change in propor3on of specific bacterial sequences for helminth and no-‐parasite groups. This measure is useful for determining the capacity of an assay to discriminate between 2 experimental groups and small effect size. The WGS method provides rich metagenomic func3onal informa3on as compared to 16S rRNA sequencing. Metagenomic func3onal informa3on for 16S rRNA reads can be inferred using PICRUST so`ware through taxonomic informa3on, but it lacks the direct evidence of genes found in WGS. On the other hand, 16S sequencing is computa3onally inexpensive, while WGS data are challenging to manage/analyse and require so`ware with complex algorithms. Our results show that WGS offers higher taxonomic resolu3on and discrimina3on along with metagenomic func3onal informa3on while 16S provides a reasonable op3on if the taxonomic informa3on is the primary focus of a study. This study provides important informa3on for selec3ng the op3mal assay based on func3on and price with implica3ons in evolu3onary inves3ga3ons and tropical medicine.
• Asymptoma3c children • Ages 4-‐6 years old • No recent an3bio3cs • qPCR and microscopy for presence of : Ascaris lumbricoides Strongyloides stercoralis Ancylostoma duodenale Giardia lamblia Necator americanus Cryptosporidium species Trichuris trichiura Entamoeba histoly;ca • NEBNext® Microbiome DNA Enrichment Kit • NEBNext® Ultra™ DNA Library Prep Kit for Illumina® • Illumina NextSeq® Whole genome sequencing • Livermore Metagenomics Analysis Toolkit (LMAT) and Diamond so`ware • Phred quality score 20 (99% base call accuracy) • Normalized to 10,000 reads for bacterial diversity
0 0.01 0.1
Proportion of sequences (%)
Proportion of sequences (%)
1
Giardia Non-Giardia
1
10
100 1000 10000
Giardia fg/µl • Greater than 1 fg/µl Giardia DNA has significant decrease in bacterial diversity to No Parasite group (p = 0.0244) 5
Giardia Helminths No Parasites Giardia Helminth
• Giardia infec3ons had lower cellular amino acid metabolic processes than helminth infec3ons (p = 0.047)
4
3
• f 2
1
• Higher Giardia burdens correlate to less bacterial diversity which could indicate worse disease status
• Giardia infected group had significant increases in Prevotella species compared to helminth groups • Coinfec3ons negated those differences • Metagenomics showed lower cellular amino acid processes and decreased cobalamin (Vitamin B12) biosynthesis genes in Giardia infected children and related to high Giardia burden
• Useful for epidemiology and morbidity studies 0 o Giardia > 1 fg/µl No Giardia Helminths • Correlate mechanism of l Giardia< 1 fg/µl Parasites Helminth decreased Vitamin B12 genes to growth delays in • High Giardia infected children • Giardia >1 fg/µl group had more children infected with had decreased cobalamin abundant Prevotella than No Parasite intes3nal parasites biosynthesis genes compared to group p = 0.037 (A) with Helminths No Parasites (p = 0.038)(A) with • Expanding understanding group decreased Prevotella to Giardia of morbidity and compensatory effects from group (p = 0.024) and Giardia/helminth malnutri3on Helminth infec3ons (p = co-‐infected nega3ng these differences 0.021)(B) B A (p = 0 .019) ( B) B • Future direc3ons: A • Correlate quan3ty of parasite DNA with clinical outcomes • Associate morbidity to changes in microbiome • Treat children with an3-‐ parasi3cs and evaluate changes in microbiome Propor3on of sequences (%)
• 99 pa3ent samples
2
Conclusions
• These findings are mostly with higher Giardia burdens and likely due to changes in intes3nal micro-‐environments due to Giardia crea3ng anaerobic microenvironments
Propor3on of sequences (%)
• Temperate climate
3
Propor3on oof f sequences sequences (%) (%) Propor3on
• Peri-‐urban community
Spearman r = -0.5491 p = 0.0244
Prevotella propor3on (%)
• Field site: Orán, Argen3na
4
Shannon Alpha Diversity
Materials and methods
• Giardia group had more anaerobic bacteria than other cohorts op3mizing condi3ons for Prevotella (p = 0.012)
• Increasing Giardia burden (fg/µl) correlates to decreasing intes3nal bacterial diversity
Prevotella propor3on (%)
• >2 billion GI parasite infec3ons worldwide in poorest and resource-‐ deprived communi3es • GI parasites may disrupt normal intes3nal microbiota • Decreased microbial biodiversity is associated with disease, including: § Malabsorp3on § Inflammatory bowel diseases • Vitamin B12 involved in metabolism of every human cell • Bacteria have the enzymes needed for vitamin B12 biosynthesis • qPCR is rapid, quan3ta3ve, high-‐ throughput and is a more reliable species-‐specific method
Results
Shannon Alpha Diversity
Introduc)on
Acknowledgements
Funding for this project was provided by the Na3onal School of Tropical Medicine, Baylor College of Medicine and New England Biolabs, Inc.
High Giardia Low Giardia No Parasites
High Giardia Low Giardia No Parasites
Giardia Helminths No Parasites Giardia High Giardia Low Giardia No Parasites Giardia Helminths No Giardia Parasites Helminth Helminth
697
