Novel approaches for bioinformatic analysis of salivary RNA sequencing data for development

Motivation Analysis of RNA sequencing (RNA-Seq) data in human saliva is challenging. Lack of standardization and unification of the bioinformatic procedures undermines saliva's diagnostic potential. Thus, it motivated us to perform this study. Results We applied principal pipelines for bioinformatic analysis of small RNA-Seq data of saliva of 98 healthy Korean volunteers including either direct or indirect mapping of the reads to the human genome using Bowtie1. Analysis of alignments to exogenous genomes by another pipeline revealed that almost all of the reads map to bacterial genomes. Thus, salivary exRNA has fundamental properties that warrant the design of unique additional steps while performing the bioinformatic analysis. Our pipelines can serve as potential guidelines for processing of RNA-Seq data of human saliva. Availability and implementation Processing and analysis results of the experimental data generated by the exceRpt (v4.6.3) small RNA-seq pipeline (github.gersteinlab.org/exceRpt) are available from exRNA atlas (exrna-atlas.org). Alignment to exogenous genomes and their quantification results were used in this paper for the analyses of small RNAs of exogenous origin. Contact dtww@ucla.edu.

[1]  Feng Li,et al.  The landscape of microRNA, Piwi-interacting RNA, and circular RNA in human saliva. , 2015, Clinical chemistry.

[2]  Makoto Murakami,et al.  Small RNA transcriptomes of two types of exosomes in human whole saliva determined by next generation sequencing. , 2013, Biological & pharmaceutical bulletin.

[3]  Juliana Vianna Pereira,et al.  Bacterial diversity in the saliva of patients with different oral hygiene indexes. , 2012, Brazilian dental journal.

[4]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[5]  Rebecca A. Reiman,et al.  Total Extracellular Small RNA Profiles from Plasma, Saliva, and Urine of Healthy Subjects , 2017, Scientific Reports.

[6]  David Elashoff,et al.  Salivary Transcriptome Diagnostics for Oral Cancer Detection , 2004, Clinical Cancer Research.

[7]  John S. Satterlee,et al.  The NIH Extracellular RNA Communication Consortium , 2015, Journal of extracellular vesicles.

[8]  N. Park,et al.  Salivary microRNA: Discovery, Characterization, and Clinical Utility for Oral Cancer Detection , 2009, Clinical Cancer Research.

[9]  John R Yates,et al.  The proteomes of human parotid and submandibular/sublingual gland salivas collected as the ductal secretions. , 2008, Journal of proteome research.

[10]  A. Vissink,et al.  Salivary proteomic and genomic biomarkers for primary Sjögren's syndrome. , 2007, Arthritis and rheumatism.

[11]  Steven L Salzberg,et al.  HISAT: a fast spliced aligner with low memory requirements , 2015, Nature Methods.

[12]  M. Tomita,et al.  Capillary electrophoresis mass spectrometry-based saliva metabolomics identified oral, breast and pancreatic cancer-specific profiles , 2009, Metabolomics.

[13]  Joel P. Brockman,et al.  The human salivary RNA transcriptome revealed by massively parallel sequencing. , 2012, Clinical chemistry.

[14]  Daniel J. Gaffney,et al.  A survey of best practices for RNA-seq data analysis , 2016, Genome Biology.

[15]  Rita R. Colwell,et al.  Microbial Community Profiling of Human Saliva Using Shotgun Metagenomic Sequencing , 2014, PloS one.

[16]  Thomas Tuschl,et al.  Comprehensive profiling of circulating microRNA via small RNA sequencing of cDNA libraries reveals biomarker potential and limitations , 2013, Proceedings of the National Academy of Sciences.

[17]  Jae Hoon Kim,et al.  Salivary transcriptomic biomarkers for detection of ovarian cancer: For serous papillary adenocarcinoma , 2012 .

[18]  A. Vissink,et al.  Preclinical validation of salivary biomarkers for primary Sjögren's syndrome , 2010, Arthritis care & research.

[19]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[20]  Rolf Apweiler,et al.  Systematic comparison of the human saliva and plasma proteomes , 2009, Proteomics. Clinical applications.

[21]  G. Illei,et al.  Deep sequencing of short RNAs reveals novel microRNAs in minor salivary glands of patients with Sjögren's syndrome. , 2012, Oral diseases.

[22]  Cole Trapnell,et al.  Ultrafast and memory-efficient alignment of short DNA sequences to the human genome , 2009, Genome Biology.

[23]  Kei-Hoi Cheung,et al.  Integration of extracellular RNA profiling data using metadata, biomedical ontologies and Linked Data technologies , 2015, Journal of extracellular vesicles.

[24]  D. Chia,et al.  Variations of oral microbiota are associated with pancreatic diseases including pancreatic cancer , 2011, Gut.

[25]  James Gimzewski,et al.  Nanostructural and Transcriptomic Analyses of Human Saliva Derived Exosomes , 2010, PloS one.

[26]  Jorge Amigo,et al.  SparkBWA: Speeding Up the Alignment of High-Throughput DNA Sequencing Data , 2016, PloS one.

[27]  D. Wong,et al.  Salivary extracellular noncoding RNA: emerging biomarkers for molecular diagnostics. , 2015, Clinical therapeutics.

[28]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[29]  David Elashoff,et al.  Serum circulating human mRNA profiling and its utility for oral cancer detection. , 2006, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[30]  Lian Hu,et al.  Identification of microRNAs predominately derived from testis and epididymis in human seminal plasma. , 2014, Clinical biochemistry.

[31]  Nagarjun Vijay,et al.  Challenges and strategies in transcriptome assembly and differential gene expression quantification. A comprehensive in silico assessment of RNA‐seq experiments , 2013, Molecular ecology.

[32]  D E Peterson,et al.  Using high throughput sequencing to explore the biodiversity in oral bacterial communities. , 2012, Molecular oral microbiology.

[33]  David Akin,et al.  Oral squamous cell carcinoma detection by salivary biomarkers in a Serbian population. , 2011, Oral oncology.

[34]  Carlo Montemagno,et al.  Interleukin 6 and interleukin 8 as potential biomarkers for oral cavity and oropharyngeal squamous cell carcinoma. , 2004, Archives of otolaryngology--head & neck surgery.

[35]  Andrew B West,et al.  RNA-Seq optimization with eQTL gold standards , 2013, BMC Genomics.

[36]  David Elashoff,et al.  Exon-level expression profiling: a comprehensive transcriptome analysis of oral fluids. , 2008, Clinical chemistry.

[37]  Mark Gerstein,et al.  Diverse human extracellular RNAs are widely detected in human plasma , 2016, Nature Communications.

[38]  Joris R Delanghe,et al.  Flow cytometry as a new method to quantify the cellular content of human saliva and its relation to gingivitis. , 2002, Clinica chimica acta; international journal of clinical chemistry.

[39]  David Elashoff,et al.  Salivary transcriptomic biomarkers for detection of resectable pancreatic cancer. , 2010, Gastroenterology.

[40]  Shanrong Zhao,et al.  A comprehensive evaluation of ensembl, RefSeq, and UCSC annotations in the context of RNA-seq read mapping and gene quantification , 2015, BMC Genomics.

[41]  David Elashoff,et al.  Electrochemical Sensor for Multiplex Biomarkers Detection , 2009, Clinical Cancer Research.

[42]  Yukie Shibata,et al.  Bacterial diversity in saliva and oral health-related conditions: the Hisayama Study , 2016, Scientific Reports.

[43]  David Elashoff,et al.  Development of transcriptomic biomarker signature in human saliva to detect lung cancer , 2012, Cellular and Molecular Life Sciences.

[44]  Waibhav Tembe,et al.  Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing. , 2013, RNA.

[45]  B. Langmead,et al.  Aligning Short Sequencing Reads with Bowtie , 2010, Current protocols in bioinformatics.

[46]  C. Thermes,et al.  Library preparation methods for next-generation sequencing: tone down the bias. , 2014, Experimental cell research.

[47]  M. Gerstein,et al.  RNA-Seq: a revolutionary tool for transcriptomics , 2009, Nature Reviews Genetics.

[48]  Lei Zhang,et al.  Proteomic Analysis of Human Saliva From Lung Cancer Patients Using Two-Dimensional Difference Gel Electrophoresis and Mass Spectrometry* , 2011, Molecular & Cellular Proteomics.

[49]  David Elashoff,et al.  Discovery and Preclinical Validation of Salivary Transcriptomic and Proteomic Biomarkers for the Non-Invasive Detection of Breast Cancer , 2010, PloS one.