Reducing costs for DNA and RNA sequencing by sample pooling using a metagenomic approach

Abstract

DNA and RNA sequencing are widely used techniques to investigate genomic modifications and gene expression. The costs for sequencing dropped dramatically in the last decade. However, due to material and labor intense steps, the sample preparation costs could not keep up with that pace. About 80% of the total costs occur prior to sequencing during DNA/RNA extraction, enrichment steps and subsequent library preparation. In this study, we investigate the potential of pooling different organisms samples prior to DNA/RNA extraction to significantly reduce costs in preparative steps. Similar to the common procedure of ligated DNA tags to pool (c)DNA samples, sequence diversity of different organisms intrinsically provide unique sequences that allow separation of reads after sequencing. With this approach, sample pooling can occur before DNA/RNA isolation and library preparation. We show that pooled sequencing of three related bacterial organisms is possible without loss of data quality at a cost reduction of approx. 50% in DNA- and RNA-seq approaches. Furthermore, we show that this approach is highly efficient down to the level of a shared genus and is, therefore, widely applicable in sequencing facilities and companies with diverse sample pools.

Ploidy in Vibrio natriegens: Very Dynamic and Rapidly Changing Copy Numbers of Both Chromosomes

“…Our results indicate that this might also be true for V. natriegens because for both chromosomes, there was a slight difference in the read numbers at 4 h, but replication had completely ceased at 5 h. (4) The ori/ter ratio was much higher for chromosome 1 than for chromosome 2 (Figure 5). This was also observed in a recent study on V. natriegens in the early exponential phase, which reported an ori/ter ratio of 5 for chromosome 1 and 2.5 for chromosome 2 [56]. This was due to the following two reasons: first, the origin copy number increase is much higher for chromosome 1 than for chromosome 2 (Figure 2), and second, the difference depends on the size of the chromosome because larger chromosomes require a longer time between initiation and termination.…”

“…If it is assumed that the ori/ter ratio might have been overestimated by 40% when using the qPCR method and all values are divided by 1.4, the highest ori/ter ratio would decrease from 9 to 6.4, a value similar to the ratio of 4.0 determined using marker frequency analysis. A recent marker frequency analysis of V. natriegens determined an ori/ter ratio of approximately five [56], which is between the two values determined in this study. Taken together, three independent analyses showed that the ori/ter ratio of V. natriegens in the early exponential phase is at least four and that multifork replication occurs.…”

Functional profile of oral plaque microbiome: Further insight into the bidirectional relationship between type 2 diabetes and periodontitis

“…In biomedical research, clinical trials often include experimental studies that present analytical problems related to small sample size; in metagenomics studies, one possible strategy to overcome this limitation is the pooling of microbiome samples before DNA amplification and metagenomic sequencing (Ray et al., 2019; Teufel & Sobetzko, 2022). Thus, our choice was to collect, for each participant, 16 samples (four subgingival plaque samples collected from four teeth and then pooled into a single specimen) by sampling subgingival plaque at sites all representative of each individual's periodontal condition, that is, for p− subjects, sampling was always performed at four sites randomly selected among those negative to bleeding on probing; in p+ patients, sampling was performed at four sites showing the deepest probing depth values among those positive to bleeding on probing.…”

Long pentraxin 3 and vitamin D receptor mRNA expression pattern of cumulus granulosa cells isolated from PCOS oocytes at different stages of nuclear maturation

“…The fold changes of VDR and PTX3 mRNA obtained from CCs at different maturation stages were compared both within and between groups. The pooling strategy of biological samples to be subjected to RNA extraction and amplification is not only cost effective, but also increases the sensitivity and specificity for the transcript to be measured [ 36 ]. For this reason, we pooled CCs collected from oocytes at the same nuclear development stage in both the PCOS and control groups by placing them in the same vials.…”