BioLiqX Small RNA-seq Kit includes all the components required to generate DNA libraries for massive parallel sequencing of small RNA (or long RNA after fragmentation) on Illumina platforms. The kit utilizes a single-tube protocol implying the addition of certain reagents and enzymes to the RNA sample in a sequential manner. The DNA library preparation workflow was originally developed for 10 ng – 1 pg inputs of highly fragmented RNA present in biofluids such as blood plasma and serum (typically between 3-100 nt). However, the kit can be also used for other inputs including total RNA purified from cells or enriched small RNA fractions of cellular RNAs.
Biological fluids (like any other sources) consist of distinct populations of RNAs carrying either hydroxyl (-OH) or phosphate (-P) groups at their 5’- and 3’- termini. Those include: (1) 5’- and 3’-phosphorylated RNAs (PP); (2) 5’-OH/3’-OH carrying RNAs (OO); (3) 5’-OH and 3’-phosphorylated RNAs (OP) and (4) 5’-phosphorylated/3’-OH RNAs (PO). For instance, the PO category includes predominantly mature miRNAs and snoRNAs. Therefore, the BioLiqX small RNA-seq kit was designed to capture RNA carrying distinct 5’- and 3’-terminal groups by including slight modifications of the protocol depending on the task and the scientific question to be addressed. The libraries generated by BioLiqX small RNA-seq kit are stranded, with Read1 and Read2 corresponding to the sense and antisense strand of the input RNA, respectively. The whole procedure can be completed within approximately 6 hours and requires a hands-on time between 20-60 minutes depending on the number of samples.
The kit is delivered in a standard cardboard microtube box that includes reagents for polynucleotide tailing and optional end-repair (Tailing Buffer, PdP Enzyme, Tailing Nucleotides, Tailing Enzyme), ligation reagents (Ligation Buffer, Ligation Enzyme), RT Mix and RT primers for reverse transcription, PreAmp Mix for the final cDNA preamplification and synthetic cel-miR-39 ssRNA control. By default, the kit is supplied with 24 standard P7 Index primers for multiplexing. The volume and exact composition of the kit (along with the primer types required for multiplexing) can be customized depending on a number of samples, type of sequencing and research goals. However, the minimal size of the kit is for 24 samples.
To avoid “Index hopping” hazard associated with some, but not all, Illumina sequencing platforms the utilization of the double (P5 and P7) indexed primers might be advised. In that cast, Heidelberg Biolabs will provide the alternative dual-index primers and other components, depending on the technology used for sequencing and the number of samples required in the multiplex.
The BioLiqX Small RNA-seq Kit is based on Capture and Amplification by Tailing and Ligation (CATL) approach to generate sequencing libraries for Illumina platforms. On the first step, single-stranded RNA is subjected to a highly efficient polyadenylation reaction. The optional steps including phosphorylation of the 5’-termini and/or removal of 3’-phosphate/cyclophosphate groups can be included depending on the experimental goals and the desired populations of RNAs to be sequenced. The polyadenylation reaction is followed by the ligation of 5’-adapter. The input RNA flanked by 5’-adapter and 3’-poly(A) tails is then converted into cDNA using anchored RT primer carrying poly(T)-rich sequence and custom 3’-adapter sequence. Multiplexing i7 indexes are introduced during the final PCR amplification with primers carrying P5 and P7 terminal sequences required for cluster generation on Illumina machines.
In most cases, the prepared library can be used directly for loading onto an Illumina sequencer after clean-up from the excess of PCR primers with magnetic SPRI beads or column-based purification. However, certain samples may require additional size-selection with either SPRI beads or the BluePippin system what secures a greater control over final library insert sizes. From our experience, the total libraries generated from cell- and cell debris-free blood plasma or serum do not require additional size-selection.
The typical experimental workflow for generating NGS libraries from plasma RNA with BioLiqX Small RNA-seq kit should include three general QC steps (Figure 1). Primarily, the quantity of input RNA could be estimated using QubitTM RNA HS Assays. However, the amounts of RNA isolated from adequate volumes of biofluids can be below the detection limit even with most sensitive methods and may range from 1 pg to 10 pg per microliter of eluate (despite a nearly complete recovery of major cell-free RNAs biotypes). As a results, it is frequently not possible to estimate optimal number of PCR cycles to be used for the amplification of the cDNA.
Figure 1. The typical experimental workflow for generating NGS libraries from plasma RNA with BioLiqX Small RNA-seq Kit including recommended QC steps.
We, therefore, recommend running aliquots of DNA libraries immediately after the final amplification step on the 3-4% Agarose gel to assess whether additional PCR cycles are required. In this example, a strong PCR product was evident after 16x amplification cycles (Figure 2A). If the library is hardly visible, 3 - 4x PCR cycles should be added before the final purification procedure. In most cases, BioLiqX libraries can be used for sequencing directly after a single SPRI beads clean-up to remove the excess of PCR primers, and the final quality check on Agilent 2200 Tapestation (Figure 2B), or a similar device. However, certain libraries may require additional size-selection steps.
Figure 2. Final DNA library prepared by BioLiqX small RNA-seq (total RNA protocol). The RNA was isolated from 400 µL of cell debris-free human blood plasma, eluted in 50 µL of RNAse-free water, and 8 µL of eluate were taken as input for library preparation. The concentration of RNA in the eluate was below the detection threshold. (A) 3% Agarose electropherogram of a 20 µL (1/5 total) aliquot of DNA library after the final 16 cycles PCR amplification. The remaining 80 µL of DNA library were purified from contaminating primers using MagSi-NGSprep Plus beads and further analyzed on Agilent 2200 TapeStation using High Sensitivity D1000 ScreenTape (B). The lower and upper peaks correspond to DNA reference markers. The peak at 177 bp corresponds to combined size of processed plasma RNA plus adapters.
After purification from an excess of primers BioLiqX small RNA-seq (three independent replicates) were sequenced on the Illumina HiSeq2000 platform. The obtained raw FASTQ files were trimmed from polyA-tails and size-selected using cutadapt software. (Figure 3)
Figure 3. The quality score reports obtained by fastqc software after HiSeq2000 sequencing of three independent BioLiqX Small RNA-seq libraries generated from human plasma RNA sample. The raw FASTQ files were trimmed using cutadapt. The reads shorter then 15 nt were discarded.
Finally, trimmed and size-selected reads were aligned to the custom curated hg38 reference transcriptomes in a sequential manner. First, all reads were mapped to RNA species with low sequence complexity and/or high number of repeats: rRNA, tRNA, RN7S, snRNA, snoRNA/scaRNA, vault RNA, RNY as well as mitochondrial chromosome (mtRNA). All reads that did not map to the above RNAs were aligned sequentially to mature miRNA, pre-miRNA , protein-coding mRNA transcripts (mRNA) and long non-coding RNAs (lncRNAs). The reads which did not map to the above RNAs were aligned to the remaining transcriptome [other ncRNAs containing mostly pseudogenes and non-protein coding parts of mRNAs]. Finally, all reads which did not map to human transcriptome were aligned to the human genome reference (rest hg38) that corresponds to introns and intergenic regions. The combined raw count tables from each replicated were combined and compared for reproducibility using correlation plots (Figure 4)
Figure 4. Correlation plots of raw human transcriptome reads obtained from three independent BioLiqX small RNA-seq libraries generated from human plasma RNA sample and sequenced on HiSeq2000.