High-scale random access on DNA storage systems

ABSTRACT Due to the rapid cost decline of synthesizing and sequencing deoxyribonucleic acid (DNA), high information density, and its durability of up to centuries, utilizing DNA as an information storage medium has received the attention of many scientists. State-of-the-art DNA storage systems exploit the high capacity of DNA and enable random access (predominantly random reads) by primers, which serve as unique identifiers for directly accessing data. However, primers come with a significant limitation regarding the maximum available number per DNA library. The number of different primers within a library is typically very small (e.g. ≈10). We propose a method to overcome this deficiency and present a general-purpose technique for addressing and directly accessing thousands to potentially millions of different data objects within the same DNA pool. Our approach utilizes a fountain code, sophisticated probe design, and microarray technologies. A key component is locality-sensitive hashing, making checks for dissimilarity among such a large number of probes and data objects feasible.

Identification of 19-bp indel of the Pleomorphic Adenoma Gene 1 in Bali cattle population

Pleomorphic adenoma gene 1 (PLAG1) is a zinc finger transcription factor gene located on bovine chromosome 14 (BTA14) affecting body size and reproduction traits in cattle. The objective of this study was to identify 19-bp indel of the PLAG1 gene in Bali cattle population. A total of 96 blood samples of Bali Cattle were collected from Balai Pembibitan Ternak Unggul dan Hijauan Pakan Ternak (BPTU-HPT) Denpasar. Genomic DNA was extracted from blood samples and used to detect 19-bp indel of the PLAG1 gene using following primer pair 5’-TCCGAACAACAGGTGAGGGAGAAAT-3’ and 5’-CCACTTCAGG-GGTGCTCTAGGTTTG-3’. The polymerase chain reaction (PCR) products using DNA pool samples were sequenced to validate the PCR product and to find out novel polymorphism in Bali cattle population. The result showed that there was no variation found in Bali cattle population based on 19-bp indel of the PLAG1 gene, which is indicated by 123 bp DNA band. However, sequence analysis of the PLAG1 gene resulted in a novel single nucleotide polymorphism (SNP) at nucleotide number 32235 of the PLAG1 gene that changed guanine (G) to adenine (A). This novel SNP could be furthermore genotyped and it might be a potential candidate marker for body size and reproduction traits in Bali cattle.

Optimizing a metabarcoding primer portfolio for species-level detection of taxa in complex mixtures of diverse fishes

DNA metabarcoding is used to enumerate and identify taxa in both environmental samples and tissue mixtures. The composition and resolution of metabarcoding data depend on the primer(s) used. Markers that amplify different genes can mitigate biases in primer affinity, amplification efficiency, and reference database resolution, but few empirical studies have evaluated markers for complementary performance. Here, we assess the individual and joint performance of 22 markers for detecting species in a DNA pool of >100 species of primarily marine and freshwater fishes, but also including representatives of elasmobranchs, cephalopods, and crustaceans. Marker performance includes the integrated effect of primer specificity and reference availability. We find that a portfolio of four markers targeting 12S, 16S, and multiple regions of COI identifies 100% of reference taxa to family and nearly 60% to species. We then use the four markers in this portfolio to evaluate metabarcoding of heterogeneous tissue mixtures, using experimental fishmeal to test: 1) the tissue input threshold to ensure detection; 2) how read depth scales with tissue abundance; and 3) the effect of non-target material in the mixture on recovery of target taxa. We consistently detect taxa that make up >1% of fishmeal mixtures and can detect taxa at the lowest input level of 0.01%, but rare taxa (<1%) were detected inconsistently across markers and replicates. Read counts showed weak correlation with tissue input, suggesting they are not a valid proxy for relative abundance. Despite this limitation, our results demonstrate the value of a primer portfolio approach—tailored to the taxa of interest—for detecting and identifying both rare and abundant species in heterogeneous tissue mixtures.

Chemistry and Bioinformatics Considerations in Using Next-Generation Sequencing Technologies to Inferring HIV Proviral DNA Genome-Intactness

HIV persists via integration of the viral DNA into the human genome. The HIV DNA pool within an infected individual is a complex population that comprises both intact and defective viral genomes, each with a distinct integration site, in addition to a unique repertoire of viral quasi-species. Obtaining an accurate profile of the viral DNA pool is critical to understanding viral persistence and resolving interhost differences. Recent advances in next-generation deep sequencing (NGS) technologies have enabled the development of two sequencing assays to capture viral near-full- genome sequences at single molecule resolution (FLIP-seq) or to co-capture full-length viral genome sequences in conjunction with its associated viral integration site (MIP-seq). This commentary aims to provide an overview on both FLIP-seq and MIP-seq, discuss their strengths and limitations, and outline specific chemistry and bioinformatics concerns when using these assays to study HIV persistence.

SNP Development in Penaeus vannamei via Next-Generation Sequencing and DNA Pool Sequencing

Next-generation sequencing and pool sequencing have been widely used in SNP (single-nucleotide polymorphism) detection and population genetics research; however, there are few reports on SNPs related to the growth of Penaeus vannamei. The purpose of this study was to call SNPs from rapid-growing (RG) and slow-growing (SG) individuals’ transcriptomes and use DNA pool sequencing to assess the reliability of SNPs. Two parameters were applied to detect SNPs. One parameter was the p-values generated using Fisher’s exact test, which were used to calculate the significance of allele frequency differences between RG and SG. The other one was the AFI (minor allele frequency imbalance), which was defined to highlight the fold changes in MAF (minor allele frequency) values between RG and SG. There were 216,015 hypothetical SNPs, which were obtained based on the transcriptome data. Finally, 104 high-quality SNPs and 96,819 low-quality SNPs were predicted. Then, 18 high-quality SNPs and 17 low-quality SNPs were selected to assess the reliability of the detection process. Here, 72.22% (13/18) accuracy was achieved for high-quality SNPs, while only 52.94% (9/17) accuracy was achieved for low-quality SNPs. These SNPs enrich the data for population genetics studies of P. vannamei and may play a role in the development of SNP markers for future breeding studies.

Detection of genetic variation and activity analysis of the promoter region of the cattle tRNA-modified gene <i>TRDMT1</i>

The tRNA modification gene in eukaryotes is relatively conservative. As an important modification gene, the TRDMT1 gene plays an important role in maintaining tRNA structural maintenance and reducing mistranslation of protein translation by methylation of specific tRNA subpopulations. Mouse and zebrafish TRDMT1 knockout experiments indicate that it may mediate growth and development through tRNA modification. However, there are no systematic reports on the function of tRNA-modified genes in livestock. In this study, Qinchuan cattle DNA pool sequencing technology was used. A G>C mutation in the −1223 bp position upstream of the TRDMT1 translation initiator codon was found. At this locus, the dual-luciferase assay indicated that different genotypes cause differences in transcriptional activity (P<0.05). Our experiment detected a natural genetic variation of a tRNA modification gene TRDMT1, which may provide potential natural molecular materials for the study of tRNA modification.

The Molecular Information About Deadwood Bacteriomes Partly Depends on the Targeted Environmental DNA

Microbiome studies mostly rely on total DNA extracts obtained directly from environmental samples. The total DNA consists of both intra- and extracellular DNA, which differ in terms of their ecological interpretation. In the present study, we have investigated for the first time the differences among the three DNA types using microbiome sequencing of Picea abies deadwood logs (Hunter decay classes I, III, and V). While the bacterial compositions of all DNA types were comparable in terms of more abundant organisms and mainly depended on the decay class, we found substantial differences between DNA types with regard to less abundant amplicon sequence variants (ASVs). The analysis of the sequentially extracted intra- and extracellular DNA fraction, respectively, increased the ecological depth of analysis compared to the directly extracted total DNA pool. Both DNA fractions were comparable in proportions and the extracellular DNA appeared to persist in the P. abies deadwood logs, thereby causing its masking effect. Indeed, the extracellular DNA masked the compositional dynamics of intact cells in the total DNA pool. Our results provide evidence that the choice of DNA type for analysis might benefit a study’s answer to its respective ecological question. In the deadwood environment researched here, the differential analysis of the DNA types underlined the relevance of Burkholderiales, Rhizobiales and other taxa for P. abies deadwood decomposition and revealed that the role of Acidobacteriota under this scenario might be underestimated, especially compared to Actinobacteriota.

Polymorphisms in JAK2 Gene are Associated with Production Traits and Mastitis Resistance in Dairy Cattle

The present study was designed to investigate the effects of single nucleotide polymorphisms (SNPs) in the JAK2 gene on the production and mastitis related traits in dairy cattle. Blood and milk samples were collected from 201 lactating dairy cattle of three breeds, i.e. Holstein Friesian (HF), Jersey (J) and Achai (A) and their crosses maintained at well-established dairy farms in Khyber Pakhtunkhwa, Pakistan. Generalized linear model was used to evaluate the association between genotypes and the studied traits. A DNA pool was made from randomly selected 30 samples which revealed three SNPs, i.e. SNP 1 in 5’ upstream region (G>A, rs379754157), SNP 2 in intron 15 (A>G, rs134192265), and SNP 3 in exon 20 (A>G, rs110298451) that were further validated in the population under study using SNaPshot technique. Of the three SNPs, SNP 1 did not obey Hardy-Weinberg equilibrium (P<0.05). SNP 2 and SNP 3 were found to be in strong linkage disequilibrium and allele G was highly prevalent compared to allele A in these SNPs. in SNP 1, the GG genotype was associated with significantly (P<0.01) higher SCC, whereas SNP 2 and SNP 3 were significantly (P<0.01) associated with higher lactose percentage compared to the other geno-types. The haplogroups association analysis revealed that H1H2 (GG GG AG) has significantly lower SCC than H2H2 (GG GG GG). The results infer that JAK2 could be an important candidate gene and the studied SNPs might be useful genetic markers for production and mastitis related traits.

Genetic variant of <i>SPARC</i> gene and its association with growth traits in Chinese cattle

SPARC is a cysteine-rich acidic secreted protein. It is a non-collagen component of bone, which is widely distributed in humans and animals and plays an important role. SPARC has been found in a variety of human cancers (breast cancer, stomach cancer, ovarian cancer, etc.) and diabetes-related research. Especially the muscle and fat metabolism are closely related. In this study, we used a DNA pool to detect a new SNP site (g.12454T > C). A total of 616 samples of four breeds of Qinchuan cattle (QC, n=176), Xianan cattle (XN, n=160), Pinan cattle (PN, n=136) and Jiaxian cattle (JX, n=144) were analyzed and identified with ARMS-PCR. In addition, we correlated SNP with growth traits and showed significant correlation with growth traits such as rump length, hip width, and body length (p<0.05). Moreover, we tested the SPARC gene expression level in different tissues belonging to XN adult cattle (n=3) and found its high expression in muscle tissues (relative to the kidney). Further, we found the SNP is able to increase the SPARC expression level in skeletal muscle (n=12). According to statistical data, this SNP site may be applied to a molecular marker of an early marker-assisted selection for early growth of beef cattle.