Navigating the hydroxymethylome: experimental biases and quality control tools for the tandem bisulfite and oxidative bisulfite Illumina microarrays

Aim: Tandem bisulfite (BS) and oxidative bisulfite (oxBS) conversion on DNA followed by hybridization to Infinium HumanMethylation BeadChips allows nucleotide resolution of 5-hydroxymethylcytosine genome-wide. Here, the authors compared data quality acquired from BS-treated and oxBS-treated samples. Materials & methods: Raw BeadArray data from 417 pairs of samples across 12 independent datasets were included in the study. Probe call rates were compared between paired BS and oxBS treatments controlling for technical variables. Results: oxBS-treated samples had a significantly lower call-rate. Among technical variables, DNA-specific extraction kits performed better with higher call rates after oxBS conversion. Conclusion: The authors emphasize the importance of quality control during oxBS conversion to minimize information loss and recommend using a DNA-specific extraction kit for DNA extraction and an oxBSQC package for data preprocessing.

Using the Packet Header Redundant Fields to Improve VoIP Bandwidth Utilization

Timeworn telecommunication are progressively being substituted by a new one that run over IP networks, which is recognized as voice over internet protocol (VoIP). VoIP has a number of qualities (e.g., inexpensive call rate), which make it progressively widespread in the telecommunication domain. However, VoIP faces plentiful obstacles that slow its growth. One of the major obstacles is poorly utilizing the network bandwidth. A number of techniques have been offered to handle this obstacle, including packet multiplexing techniques. This paper designs an original multiplexing techniques, called packet multiplexing and carrier header (PM-CH), to decrease the quantity of the bandwidth consumed by VoIP. PM-CH protect the bandwidth by multiplexing the packets in a header and using the Timestamp field in the RTP header. The achievement of the PM-CH technique was examined depends on connection capacity and payload shortening. Simulation outcomes show that the PM-CH technique outperforms the contrast technique in the two factors. For instance, the PM-CH technique’s connection capacity outperforms the comparable technique by 58.9% when the connection bandwidth is 1000 kbps. Consequently, the PM-CH technique attains its objective of reducing the unexploited bandwidth caused by VoIP.

Vocal communication in wild chimpanzees: a call rate study

Background Patterns of vocal communication have implications for species conservation: a change in calling behaviour can, for instance, reflect a disturbed habitat. More importantly, call rate is a parameter that allows conservation planners to convert call density into animal density, when detecting calls with a passive acoustic monitoring system (PAM). Methods We investigated chimpanzee (Pan troglodytes schweinfurthii) call rate during the late dry season in the Issa Valley, western Tanzania by conducting focal follows. We examined the socio-ecological factors that influence call production rate of savanna woodland chimpanzees. Results We found that sex, proportion of time spent in a vegetation type, proportion of time spent travelling, time of the day, party size and swollen parous female presence had a significant effect on the call rate. Call rate differed among the different demographic classes with subadult and adult males vocalising twice as often as the subadult and adult females and three times as often as the juveniles. Applications The use of PAM and recent statistical developments to estimate animal density is promising but relies on our knowing individual call rate, often not available for many species. With the improvement in automatic call detection, we anticipate that PAM will increasingly be broadly applied to primates but also across taxa, for conservation.

Intra-Group Orca Call Rate Modulation Estimation Using Compact Four Hydrophones Array

Acoustic emissions are vital for orca (Orcinus orca) socializing, hunting, and maintaing spatial awareness. Studying the acoustic emissions of orcas on an individual basis often results in interference with their natural behaviors through mounting tags or following by boat. In order to analyze their inter- and intra-group communication, we propose a study allowing us to associate vocalizations with their emitter (matriline and when possible individual). Such a non-interfering device for allocating calls to individual orcas could substantially boost our understanding of their complex acoustic world. Our experimental protocol was based on a compact array of four hydrophones fixed near the shore, operable up to 1 km away from the path of orcas. It was used during summer 2019 at the research station OrcaLab, northern Vancouver Island, Canada. A total of 722 calls were extracted, jointly with visual identification and azimuth of surfacing orcas, allowing validation of the acoustic diarization and azimuth estimations of the orca calls. We then calculated the Call Rate (CR) for each matriline or when possible individual in order to describe their acoustic activity. Preliminary results show that CR could be modulated according to the distance of the signaler from a group, the presence of another group, or anthropic pressure.

The Impact of Radiologist Screening Mammogram Reading Volume on Performance in the Ontario Breast Screening Program

Purpose: Although some studies have shown increasing radiologists’ mammography volumes improves performance, there is a lack of evidence specific to digital mammography and breast screening program performance targets. This study evaluates the relationship between digital screening volume and meeting performance targets. Methods: This retrospective cohort study included 493 radiologists in the Ontario Breast Screening Program who interpreted 1,762,173 screening mammograms in participants ages 50-90 between 2014 and 2016. Associations between annual screening volume and meeting performance targets for abnormal call rate, positive predictive value (PPV), invasive cancer detection rate (CDR), sensitivity, and specificity were modeled using mixed-effects multivariate logistic regression. Results: Most radiologists read 500-999 (36.7%) or 1,000-1,999 (31.0%) screens annually, and 18.5% read ≥2,000. Radiologists who read ≥2,000 annually were more likely to meet abnormal call rate (OR = 3.85; 95% CI: 1.17-12.61), PPV (OR = 5.36; 95% CI: 2.53-11.34), invasive CDR (OR = 4.14; 95% CI: 1.50-11.46), and specificity (OR = 4.07; 95% CI: 1.89-8.79) targets versus those who read 100-499 screens. Radiologists reading 1,000-1,999 screens annually were more likely to meet PPV (OR = 2.32; 95% CI: 1.22-4.40), invasive CDR (OR = 3.36; 95% CI: 1.49-7.59) and specificity (OR = 2.00; 95% CI: 1.04-3.84) targets versus those who read 100-499 screens. No significant differences were observed for sensitivity. Conclusions: Annual reading volume requirements of 1,000 in Canada are supported as screening volume above 1,000 was strongly associated with achieving performance targets for nearly all measures. Increasing the minimum volume to 2,000 may further reduce the potential limitations of screening due to false positives, leading to improvements in overall breast screening program quality.

COPILOT: a Containerised wOrkflow for Processing ILlumina genOtyping daTa

Background: The Illumina genotyping microarrays generate data in image format, which is processed by the platform-specific software GenomeStudio, followed by an array of complex bioinformatics analyses. This process can be time-consuming, lead to reproducibility errors, and be a daunting task for novice bioinformaticians. Results: Here we introduce the COPILOT (Containerised wOrkflow for Processing ILlumina genOtyping daTa) protocol, which provides an in-depth and clear guide to process raw Illumina genotype data in GenomeStudio, followed by a containerised workflow to automate an array of complex bioinformatics analyses involved in a GWAS quality control (QC). The COPILOT protocol was applied to two independent cohorts consisting of 2791 and 479 samples genotyped on the Infinium Global Screening (GSA) array with Multi-disease (MD) drop-in (~750,000 markers) and the Infinium H3Africa consortium array (~2,200,000 markers) respectively. Following the COPILOT protocol, an average sample quality improvement of 1.24% was observed across sample call rates, with notable improvement for low-quality samples. For example, from the 3270 samples processed, 141 samples had an initial sample call rate below 98%, averaging 96.6% (95% CI 95.6-97.7%), which is considered below the acceptable sample call rate threshold for a typical GWAS analysis. However, following the COPILOT protocol, all 141 samples had a call rate above 98% after QC and averaged 99.6% (95% CI 99.5-99.7%). In addition, the COPILOT pipeline automatically identified potential data issues, including gender discrepancies, heterozygosity outliers, related individuals, and population outliers through ancestry estimation. Conclusions: The COPILOT protocol makes processing Illumina genotyping data transparent, effortless and reproducible. The container is deployable on multiple platforms, improves data quality, and the end product is analysis-ready PLINK formatted data, with a comprehensive and interactive summary report to guide the user for further data analyses.

Discomfort-related changes of call rate and acoustic variables of ultrasonic vocalizations in adult yellow steppe lemmings Eolagurus luteus

Potential of ultrasonic vocalizations (USVs) to reflect a degree of discomfort of a caller is mostly investigated in laboratory rats and mice but poorly known in other rodents. We examined 36 (19 male, 17 female) adult yellow steppe lemmings Eolagurus luteus for presence of USVs during 8-min experimental trials including 2-min test stages of increasing discomfort: isolation, touch, handling and body measure. We found that 33 of 36 individuals vocalized at isolation stage, i.e., without any human impact. For 14 (6 male and 8 female) individuals, a repeated measures approach revealed that increasing discomfort from isolation to handling stages resulted in increase of call power quartiles and fundamental frequency, whereas call rate remained unchanged. We discuss that, in adult yellow steppe lemmings, the discomfort-related changes of USV fundamental frequency and power variables follow the same common rule as the audible calls of most mammals, whereas call rate shows a different trend. These data contribute to research focused on searching the universal acoustic cues to discomfort in mammalian USVs.

Call rate in Common Cuckoos does not predict body size and responses to conspecific playbacks

The brood parasitic Common Cuckoo Cuculus canorus is best known for its two-note “cu-coo” call which is almost continuously uttered by male during the breeding season and can be heard across long distances in the field. Although the informative value of the cuckoo call was intensively investigated recently, it is still not clear whether call characteristic(s) indicate any of the phenotypic traits of the respective vocalising individuals. To fill this gap, we studied whether the call rate of male cuckoos (i.e., the number of calls uttered per unit of time) provides information on their body size, which might be a relevant trait during intrasexual territorial conflicts. We captured free-living male cuckoos and measured their body size parameters (mass, wing, tail and tarsus lengths). Each subject was then radio-tagged, released, and its individual “cu-coo” calls were recorded soon after that in the field. The results showed that none of the body size parameters covaried statistically with the call rates of individual male Common Cuckoos. In addition, we experimentally tested whether the “cu-coo” call rates affect behavioural responses of cuckoos using playbacks of either a quicker or a slower paced call than the calls with natural rates. Cuckoos responded similarly to both types of experimental playback treatments by approaching the speaker with statistically similar levels of responses as when presented with calls at the natural rate. We conclude that male Common Cuckoos do not advertise reliable information acoustically regarding their body size, and so, cuckoo calls are neither useful to characterize cuckoos’ phenotypic traits directly nor to indicate environmental quality indirectly.

Human sample authentication in biomedical research: comparison of two platforms

Samples used in biomedical research are often collected over years, in some cases from subjects that may have died and thus cannot be retrieved in any way. The value of these samples is priceless. Sample misidentification or mix-up are unfortunately common problems in biomedical research and can eventually result in the publication of incorrect data. Here we have compared the Fluidigm SNPtrace and the Agena iPLEX Sample ID panels for the authentication of human genomic DNA samples. We have tested 14 pure samples and simulated their cross-contamination at different percentages (2%, 5%, 10%, 25% and 50%). For both panels, we report call rate, allele intensity/probability score, performance in distinguishing pure samples and contaminated samples at different percentages, and sex typing. We show that both panels are reliable and efficient methods for sample authentication and we highlight their advantages and disadvantages. We believe that the data provided here is useful for sample authentication especially in biorepositories and core facility settings.

RNA-Seq Data for Reliable SNP Detection and Genotype Calling: Interest for Coding Variant Characterization and Cis-Regulation Analysis by Allele-Specific Expression in Livestock Species

In addition to their common usages to study gene expression, RNA-seq data accumulated over the last 10 years are a yet-unexploited resource of SNPs in numerous individuals from different populations. SNP detection by RNA-seq is particularly interesting for livestock species since whole genome sequencing is expensive and exome sequencing tools are unavailable. These SNPs detected in expressed regions can be used to characterize variants affecting protein functions, and to study cis-regulated genes by analyzing allele-specific expression (ASE) in the tissue of interest. However, gene expression can be highly variable, and filters for SNP detection using the popular GATK toolkit are not yet standardized, making SNP detection and genotype calling by RNA-seq a challenging endeavor. We compared SNP calling results using GATK suggested filters, on two chicken populations for which both RNA-seq and DNA-seq data were available for the same samples of the same tissue. We showed, in expressed regions, a RNA-seq precision of 91% (SNPs detected by RNA-seq and shared by DNA-seq) and we characterized the remaining 9% of SNPs. We then studied the genotype (GT) obtained by RNA-seq and the impact of two factors (GT call-rate and read number per GT) on the concordance of GT with DNA-seq; we proposed thresholds for them leading to a 95% concordance. Applying these thresholds to 767 multi-tissue RNA-seq of 382 birds of 11 chicken populations, we found 9.5 M SNPs in total, of which ∼550,000 SNPs per tissue and population with a reliable GT (call rate ≥ 50%) and among them, ∼340,000 with a MAF ≥ 10%. We showed that such RNA-seq data from one tissue can be used to (i) detect SNPs with a strong predicted impact on proteins, despite their scarcity in each population (16,307 SIFT deleterious missenses and 590 stop-gained), (ii) study, on a large scale, cis-regulations of gene expression, with ∼81% of protein-coding and 68% of long non-coding genes (TPM ≥ 1) that can be analyzed for ASE, and with ∼29% of them that were cis-regulated, and (iii) analyze population genetic using such SNPs located in expressed regions. This work shows that RNA-seq data can be used with good confidence to detect SNPs and associated GT within various populations and used them for different analyses as GTEx studies.