Preparation of Labeled DNA, RNA, and Oligonucleotide Probes

Labeled nucleic acids and oligonucleotides are typically generated by enzymatic methods such as end-labeling, random priming, nick translation, in vitro transcription, and variations of the polymerase chain reaction (PCR). Some of these methods place the label in specific locations within the nucleic acid (e.g., at the 5′ or 3′ terminus); others generate molecules that are labeled internally at multiple sites. Some methods yield labeled single-stranded products, whereas others generate double-stranded nucleic acids. Finally, some generate probes of defined length, whereas others yield a heterogeneous population of labeled molecules. Options available for generating and detecting labeled nucleic acids, as well as advice on designing oligonucleotides for use as probes, is included here.

A random priming amplification method for whole genome sequencing of SARS-CoV-2 and H1N1 influenza A virus.

Background: Non-targeted whole genome sequencing is a powerful tool to comprehensively identify constituents of microbial communities in a sample. There is no need to direct the analysis to any identification before sequencing which can decrease the introduction of bias and false negatives results. It also allows the assessment of genetic aberrations in the genome (e.g., single nucleotide variants, deletions, insertions and copy number variants) including in noncoding protein regions. Methods: The performance of four different random priming amplification methods to recover RNA viral genetic material of SARS-CoV-2 were compared in this study. In method 1 (H-P) the reverse transcriptase (RT) step was performed with random hexamers whereas in methods 2-4 RT incorporating an octamer primer with a known tag. In methods 1 and 2 (K-P) sequencing was applied on material derived from the RT-PCR step, whereas in methods 3 (SISPA) and 4 (S-P) an additional amplification was incorporated before sequencing. Results: The SISPA method was the most effective and efficient method for non-targeted/random priming whole genome sequencing of COVID that we tested. The SISPA method described in this study allowed for whole genome assembly of SARS-CoV-2 and influenza A(H1N1)pdm09 in mixed samples. We determined the limit of detection and characterization of SARS-CoV-2 virus which was 103 pfu/ml (Ct, 22.4) for whole genome assembly and 101 pfu/ml (Ct, 30) for metagenomics detection. Conclusions: The SISPA method is predominantly useful for obtaining genome sequences from RNA viruses or investigating complex clinical samples as no prior sequence information is needed. It might be applied to monitor genomic virus changes, virus evolution and can be used for fast metagenomics detection or to assess the general picture of different pathogens within the sample.

Highly efficient single-stranded DNA ligation technique improves low-input whole-genome bisulfite sequencing by post-bisulfite adaptor tagging

Whole-genome bisulfite sequencing (WGBS) is the current gold standard of methylome analysis. Post-bisulfite adaptor tagging (PBAT) is an increasingly popular WGBS protocol because of high sensitivity and low bias. PBAT originally relied on two rounds of random priming for adaptor-tagging of single-stranded DNA (ssDNA) to attain high efficiency but at a cost of library insert length. To overcome this limitation, we developed terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA (TACS) ligation as an alternative to random priming. In this method, TdT attaches adenylates to the 3′-end of input ssDNA, which are then utilized by RNA ligase as an efficient connector to the ssDNA adaptor. A protocol that uses TACS ligation instead of the second random priming step substantially increased the lengths of PBAT library fragments. Moreover, we devised a dual-library strategy that splits the input DNA to prepare two libraries with reciprocal adaptor polarity, combining them prior to sequencing. This strategy ensured an ideal base–color balance to eliminate the need for DNA spike-in for color compensation, further improving the throughput and quality of WGBS. Adopting the above strategies to the HiSeq X Ten and NovaSeq 6000 platforms, we established a cost-effective, high-quality WGBS, which should accelerate various methylome analyses.

Isolation of differentially expressed genes involved in clubroot disease

The interaction between Plasmodiophora brassicae and its host Brassica rapa is investigated by two strategies. (1) IAA-conjugate hydrolases: Root hypertrophy in club root disease is dependent on increased auxin levels and these could result from auxin-conjugate hydrolysis. So far we isolated 5 different cDNA fragments out of various tissues which revealed high identity to IAR3/ILL5, ILL2, ILL3, ILL6 and ILR1 genes from Arabidopsis by comparison with database entries. (2) Random priming: Using this method, we have so far obtained 26 clones from clubroot tissue, from which several sequences may be components of plant signal transduction chains, metabolic pathways and transcriptional regulation.

A Comparison of mRNA Sequencing with Random Primed and 3’-Directed Libraries

Deep mRNA sequencing (mRNAseq) is the state-of-the-art for whole transcriptome measurements. A key step is creating a library of cDNA sequencing fragments from RNA. This is generally done by random priming, creating multiple sequencing fragments along the length of each transcript. A 3’ end-focused library approach cannot detect differential splicing, but has potentially higher throughput at lower cost (~10-fold lower), along with the ability to improve quantification by using transcript molecule counting with unique molecular identifiers (UMI) to correct for PCR bias. Here, we compare implementation of such a 3’-digital gene expression (3’-DGE) approach with “conventional” random primed mRNAseq, which has not yet been done. We find that while conventional mRNAseq detects ~15% more genes, the resulting lists of differentially expressed genes and therefore biological conclusions and gene signatures are highly concordant between the two techniques. We also find good quantitative agreement on the level of individual genes between the two techniques in terms of both read counts and fold change between two conditions. We conclude that for high-throughput applications, the potential cost savings associated with the 3’-DGE approach are a very reasonable tradeoff for modest reduction in sensitivity and inability to observe alternative splicing, and should enable much larger scale studies focused on not only differential expression analysis, but also quantitative transcriptome profiling. The computational scripts and programs, along with experimental standard operating procedures used in our pipeline presented here, are freely available on our website (www.dtoxs.org).

151 COMPARATIVE ANALYSIS OF FRESH AND CRYOPRESERVED BOAR SPERMATOZOA USING RNA SEQUENCING

Fertility of cryopreserved spermatozoa is significantly reduced compared with that of their fresh counterparts, which is certainly due to the inflicted sublethal damage to spermatozoa that is observed at various molecular and cellular levels. The identification and characterisation of this damage will help us better understand sperm cryobiology and therefore develop suitable media and procedures to improve sperm cryopreservation and fertility outcomes, especially in swine. Here, we present our preliminary assessment of RNA pools of fresh and frozen‐thawed spermatozoa using RNA-sequencing technology. Semen ejaculates of 8 fertile boars were harvested and divided into 2 fractions for each ejaculate. Fraction 1 was freshly extended in commercial diluent (FD) and fraction 2 was frozen in 5-mL plastic straws (FT). Both specimens were shipped to our laboratory for analyses. The samples were purified through Percoll gradient centrifugation and resulting motile spermatozoa were washed in cold PBS. Pelleted spermatozoa were used for total RNA extraction, followed by an in-column DNase digestion. Purity and integrity of RNA samples were checked and rRNA depleted. After random priming, 40 million short cDNA reads were produced using Illumina RNA-Seq technology (Illumina Inc., San Diego, CA, USA). All reads were aligned to the pig reference genome and the produced genome-scale transcription maps consisted of both the transcript structure and the expression level of each gene mapped. Analysis of FD sperm RNA revealed a total of 18 357 sequence tags that were successfully mapped to all pig chromosomes and the mitochondrial genome. Frozen‐thawed spermatozoa showed only 16 864 sequence tags. In both FD and FT samples, chromosomes 1, 2, 6, 7, and 13 contained, in total, the highest density of mapped transcripts (>42%). Chromosome Y and mitochondrial RNAs had the lowest sequence tags mapped (<0.08%). A comparative analysis of FD and FT datasets revealed a net decrease in the total number of sequence tags (1493) with each chromosome being affected, except mitochondria. Chromosomes of FT samples showed a strong (>10%; 17, 7, 4, Y, and X) to moderate (10 to 5%) or weak (≤5%) reduction in RNA numbers. Structural annotation revealed a diverse population of sperm transcripts comprising both coding (mRNA) and noncoding (rRNA, snRNA, and mtRNA) RNAs. In both FD and FT samples, noncoding RNAs were among the most abundant sequence tags. Approximately 12 355 of sequence tags in FD v. 10 948 in FT spermatozoa were annotated with ENSEMBL and the selected genes are under investigation for comparative analyses using RT-PCR. In conclusion, mature boar spermatozoa contain a large pool of coding and non-coding RNAs that can be affected by the freezing-thawing procedure. Inflicted damage affects RNAs of all chromosomes with a great effect being seen on chromosome X. Generated datasets have the potential to lead to further study of the cryo-damage associated with reduced fertility of cryopreserved spermatozoa. Study was supported by USDA-ARS Biophotonics initiative grant # 58-6402-3-0120 and MAFES-SRI grants.