ISEV position paper: extracellular vesicle RNA analysis and bioinformatics

Figures & data

Table I Small-RNA-sequencing library preparation: most widely used library construction methods

Method	Step 1	Step 2	Step 3	Step 4
Hybridization method (e.g. SREK for SOLiD)	Two double-stranded adapters that contain degenerate 5′- or 3′-end overhangs	Ligation adapters with T4 RNA ligase	Reverse transcription into cDNA	Further amplification by PCR using primers that anneal to both adapter sequences
Polyadenylation based (A)	Addition 3′ poly A tail using Poly (A) polymerase	Ligation RNA adapter to the 5′ end using T4 RNA ligase	Reverse transcription into cDNA	Further amplification by PCR
Polyadenylation based (B)	Addition 3′ poly A tail using Poly (A) polymerase	Reverse transcription into cDNA	Ligation adapter to the 5′ end using T4 RNA ligase 1	Further amplification by PCR
Sequential adapter ligation (e.g. Illumina) (A)	Sequential ligation of 3′- and 5′-adapter oligonucleotides directly to small RNA	Ligation RNA adapter to the 5′ end using T4 RNA ligase	Reverse transcription into cDNA	Further amplification by PCR
Sequential adapter ligation (e.g. Illumina) (B)	Sequential ligation of 3′- and 5′-adapter oligonucleotides directly to small RNA	Reverse transcription into cDNA	Ligation adapter to the 5′ end using T4 RNA ligase 1	Further amplification by PCR

Table II Parameters with regard to sRNA library preparation that should be considered

Parameter	Effects to be considered	Approaches
RNA structures	Some 5′- or 3′-end modifications of RNA sequences are not reactive or have reduced reactivity for enzymatic steps (adapter ligations) involved in library preparation. Sequences of small RNAs and their secondary and tertiary self-structures can affect the efficiency of 3′- and 5′-adapter ligation (34).	Enzymatic treatments can be used to convert small RNAs of interest to have appropriate and homogenous ends in order to be ligated to adapters with good efficiency (35).
RNA size distribution	Potential transcript length bias	Selection of small RNAs of specific sizes increases sequencing depth for sequences of interest.
Library construction method	Different barcode sequences can influence the RNA and adapter cofold structures, which may cause changes in ligation efficiency (36) Occurrence of adapter dimers	Use barcodes positioned in primers during library pre-amplification (37) or post-amplification barcode strategies (38). Removal of adapter dimers improves sequencing of specific products; removal of non-productive sequencing reads.

Table III Checklist experimental details for publication

Step	Parameters to be described
Cell culture	Cell type Confluency Use of vesicle-depleted serum or serum-free medium Cell viability Mycoplasma test
EV isolation	Differential centrifugation steps Filtration steps Density gradient Commercially available kits (e.g. chemical, column based)
evRNA sample preparation	RNase/DNase treatment of EVs Use of RNA spike RNA isolation method/kit Bioanalyzer profile of evRNA
Library preparation	Enzymatic treatment to remove phosphates, caps, etc. Method/kit for ligating adapters Pre-amplification steps Size selection steps Bioanalyzer profile of library
Sequencing	Platform Maximum read length Direction (paired or single end) Number of cycles Number of replicates (biological or technical)
Bioinformatics	Pre-processing software (trimming/clipping, cut-off values) Reference genome assemblies (release numbers) Primary analysis software (alignment and mapping criteria) Browsers and annotation tools Normalization methods and software Statistical methods and tools for differential expression analysis
Validation	Validation technique Normalization procedure
Deposition in database	Name of database

Hafner M, Renwick N, Brown M, Mihailovic A, Holoch D, Lin C, etal. RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries. RNA. 2011; 17: 1697–712.

 PubMed Web of Science ®Google Scholar

Zhuang F, Fuchs RT, Sun Z, Zheng Y, Robb GB. Structural bias in T4 RNA ligase-mediated 3′-adapter ligation. Nucleic Acids Res. 2012; 40: e54.

 PubMed Web of Science ®Google Scholar

Alon S, Vigneault F, Eminaga S, Christodoulou DC, Seidman JG, Church GM, etal. Barcoding bias in high-throughput multiplex sequencing of miRNA. Genome Res. 2011; 21: 1506–11.

 PubMed Web of Science ®Google Scholar

Buermans HP, Ariyurek Y, van Ommen G, den Dunnen JT, t Hoen PA. New methods for next generation sequencing based microRNA expression profiling. BMC Genom. 2010; 11: 716.

 Web of Science ®Google Scholar

Van Nieuwerburgh F, Soetaert S, Podshivalova K, Ay-Lin Wang E, Schaffer L, Deforce D, etal. Quantitative bias in Illumina TruSeq and a novel post amplification barcoding strategy for multiplexed DNA and small RNA deep sequencing. PLoS One. 2011; 6: e26969.

 PubMed Web of Science ®Google Scholar