Comparison of DNA and RNA sequencing of total nucleic acids from human cervix for metagenomics

Laila Sara Arroyo Mühr,Joakim Dillner,Agustin Enrique Ure,Karin Sundström,Emilie Hultin

doi:10.1038/s41598-021-98452-4

Laila Sara Arroyo Mühr, Joakim Dillner + Show 3 more

Open Access

https://doi.org/10.1038/s41598-021-98452-4

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Abstract

Although metagenomics and metatranscriptomics are commonly used to identify bacteria and viruses in human samples, few studies directly compare these strategies. We wished to compare DNA and RNA sequencing of bacterial and viral metagenomes and metatranscriptomes in the human cervix. Total nucleic acids from six human cervical samples were subjected to DNA and RNA sequencing. The effect of DNase-treatment before reverse transcription to cDNA were also analyzed. Similarities and differences in the metagenomic findings with the three different sequencing approaches were evaluated. A higher proportion of human sequences were detected by DNA sequencing (93%) compared to RNA sequencing without (76%) and with prior DNase-treatment (11%). On the contrary, bacterial sequences increased 17 and 91 times. However, the number of detected bacterial genera were less by RNA sequencing, suggesting that only a few contribute to most of the bacterial transcripts. The viral sequences were less by RNA sequencing, still twice as many virus genera were detected, including some RNA viruses that were missed by DNA sequencing. Metatranscriptomics of total cDNA provided improved detection of mainly transcribed bacteria and viruses in cervical swabs as well as detection of RNA viruses, compared to metagenomics.

Highlights

The number of bacterial cells within the human body is approximately the same as the number of human cells, 1013 in total[1], and there are approximately 150 times more genes in the human microbiome compared to the human genome[2,3]
Several disadvantages might exist when trying to enrich viruses in a biospecimen: (a) unknown viruses not targeted with the enrichment approach might escape identification, (b) a proportion of viruses might be lost depending on the enrichment method, (c) if the human genome is depleted in the sample in order to enrich for viruses, information regarding the human genome will not be included and possible viral integration may fall undetected and, (d) RNA viruses will not be identified when using a DNA extraction/DNA sequencing approach
The annotated bacterial reads increased in relative frequency as well as in total number of reads by RNA sequencing compared to DNA sequencing (Fig. 1a)

Summary

Introduction

The number of bacterial cells within the human body is approximately the same as the number of human cells, 1013 in total[1], and there are approximately 150 times more genes in the human microbiome compared to the human genome[2,3]. Viruses do not share any specific gene or other DNA/RNA sequence and other sequencing approaches than amplicon sequencing are necessary to identify and study them. Different viral target enrichment methods like filtration can be used prior massively parallel DNA sequencing in order to study the virome[6]. One approach to study the whole virome (as well as the human genome) is based on sequencing all the DNA present in a specimen, so called shotgun DNA s equencing[7,8,9]. To shotgun DNA sequencing, massively parallel cDNA sequencing from total RNA can be used to explore the metatranscriptomes within a specimen in order to perform microbiome and virome profiling[10] in addition to analysis for differential gene expression of both human, microbial and viral genes. The vaginal microbiota may have an important role to protect against harmful infections like for instance HPVs, the cervical microbiota may serve as a potential biomarker for cancer progression r isk[17,20,21]

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