Accurate single-cell genotyping utilizing information from the local genome territory

Single-nucleotide variant (SNV) detection in the genome of single cells is affected by DNA amplification artefacts, including imbalanced alleles and early PCR errors. Existing single-cell genotyper accuracy often depends on the quality and coordination of both the target single-cell and external data, such as heterozygous profiles determined by bulk data. In most single-cell studies, information from different sources is not perfectly matched. High-accuracy SNV detection with a limited single data source remains a challenge. We developed a new variant detection method, SCOUT (Single Cell Genotyper Utilizing Information from Local Genome Territory), the greatest advantage of which is not requiring external data while base calling. By leveraging base count information from the adjacent genomic region, SCOUT classifies all candidate SNVs into homozygous, heterozygous, intermediate and low major allele SNVs according to the highest likelihood score. Compared with other genotypers, SCOUT improves the variant detection performance by 2.0–77.5% in real and simulated single-cell datasets. Furthermore, the running time of SCOUT increases linearly with sequence length; as a result, it shows 400% average acceleration in operating efficiency compared with other methods.

Deep splicing plasticity of the human adenovirus type 5 transcriptome as a driver of virus evolution

Viral genomes are characterised by having high gene density and complex transcription strategies. One of the most complex is adenovirus which has a double stranded DNA genome and is the archetypal viral system in which splicing was first discovered. Understanding the transcriptional landscape using conventional mRNA cloning or more recent Illumina-based deep sequencing methods offers insight but also has limitations, including the potential for reverse transcription or PCR amplification artefacts and bias. Here we used direct RNA long read length sequencing on an Oxford Nanopore MinION device to gain a quantitative system-wide overview of transcription and splicing as it dynamically changes during a human adenovirus type 5 infection. This global overview revealed an extensive and hitherto unappreciated complexity of alternative splicing and secondary initiating codon usage. Allied to this, analysis of viral polyadenylation patterns over time showed that most viral transcripts tended to shorter polyadenylation lengths as the infection progressed. Moreover, development and use of an ORF-centric bioinformatics pipeline for analysis of sequenced mRNA, provided both a quantitative and deeper qualitative understanding of the genetic potential of this virus. The data strikingly illustrated that across the viral genome adenovirus made multiple distinctly spliced transcripts that coded for the same ORF. Indeed, as many as 11,000 different splicing patterns were recorded across the viral genome over the three time points analysed. This constitutive low level use of alternative splicing patterns and secondary ORFs potentially enables the virus to maximise its coding potential over evolutionary timescales.

Redescription of the advertisement call of Brachycephalus tridactylus (Anura: Brachycephalidae)

Background. Brachycephalus includes miniaturized frogs with restricted geographical distributions throughout the Brazilian Atlantic Forest. Ecological data for most species are still scarce. For instance, advertisement calls have only been described for 12 of the 36 known species, including B. tridactylus, a recently described species from southern Brazil. Posteriorly, features of the advertisement call of B. tridactylus were compared with congeners and the unique characteristics of its call were highlighted. To confirm these potentially divergent characteristics, we reanalysed an original recording of B. tridactylus and analysed our own recordings and verified that the original description of its advertisement call is inaccurate. Thus, we redescribe its advertisement calls. Methods. We asked the descriptors of B. tridactylus the original recordings that they made and requested access to the only original recording deposited in a collection of sounds. We received from André Lima a copy of one recording, the same as the one that had deposited, and obtained permission to re-analyze it. We studied this recording and compared it with our own recordings, made at the type locality of the species on March, 2016. Sound samples were analysed with Raven Pro 1.5.0 and call analyses were made under a note-centered approach. Results. The original recording was amplified somehow by at least 6 dB and was also clearly low-pass filtered with a cutoff frequency of 10 kHz. Our analyses did not allow us to recognize several of the acoustic parameters normally described in Brachycephalus. The sound we heard from the notes overlapped with other signals (noise?), which prevented us from clearly determining the end of the note and other important features, such as the presence of pulses. According to our recordings (n = 15 individuals), B. tridactylus emitted a relatively long advertisement call (50.8 s, on average), composed by 10–13 notes emitted in a note rate of 3.7–8.3 notes per minute. Only isolated notes were present. The notes were composed by 1–3 pulses and the note duration varied from 0.002–0.021 s. Discussion. The original description of the call of B. tridactylus is incorrect because it included background noise and amplification artefacts as part of the call parameters. However, we recognize that the original recording and our recordings have captured the same type of call. In our measurements of the species calls, note duration was nearly an order of magnitude shorter as the original description. The existence of notes with 1–3 pulses was not acknowledged in the original description. With few pulses per notes, the advertisement call of B. tridactylus is distinct from the notes with several pulses of B. ephippium, B. pitanga, B. crispus, B. sulfuratus, and B. darkside. The advertisement calls of B. tridactylus is also distinct from that of B. albolineatus and B. mirissimus by having only isolates notes, instead of isolated notes and note groups.

Redescription of the advertisement call of Brachycephalus tridactylus (Anura: Brachycephalidae)

Background. Brachycephalus includes miniaturized frogs with restricted geographical distributions throughout the Brazilian Atlantic Forest. Ecological data for most species are still scarce. For instance, advertisement calls have only been described for 12 of the 36 known species, including B. tridactylus, a recently described species from southern Brazil. Posteriorly, features of the advertisement call of B. tridactylus were compared with congeners and the unique characteristics of its call were highlighted. To confirm these potentially divergent characteristics, we reanalysed an original recording of B. tridactylus and analysed our own recordings and verified that the original description of its advertisement call is inaccurate. Thus, we redescribe its advertisement calls. Methods. We asked the descriptors of B. tridactylus the original recordings that they made and requested access to the only original recording deposited in a collection of sounds. We received from André Lima a copy of one recording, the same as the one that had deposited, and obtained permission to re-analyze it. We studied this recording and compared it with our own recordings, made at the type locality of the species on March, 2016. Sound samples were analysed with Raven Pro 1.5.0 and call analyses were made under a note-centered approach. Results. The original recording was amplified somehow by at least 6 dB and was also clearly low-pass filtered with a cutoff frequency of 10 kHz. Our analyses did not allow us to recognize several of the acoustic parameters normally described in Brachycephalus. The sound we heard from the notes overlapped with other signals (noise?), which prevented us from clearly determining the end of the note and other important features, such as the presence of pulses. According to our recordings (n = 15 individuals), B. tridactylus emitted a relatively long advertisement call (50.8 s, on average), composed by 10–13 notes emitted in a note rate of 3.7–8.3 notes per minute. Only isolated notes were present. The notes were composed by 1–3 pulses and the note duration varied from 0.002–0.021 s. Discussion. The original description of the call of B. tridactylus is incorrect because it included background noise and amplification artefacts as part of the call parameters. However, we recognize that the original recording and our recordings have captured the same type of call. In our measurements of the species calls, note duration was nearly an order of magnitude shorter as the original description. The existence of notes with 1–3 pulses was not acknowledged in the original description. With few pulses per notes, the advertisement call of B. tridactylus is distinct from the notes with several pulses of B. ephippium, B. pitanga, B. crispus, B. sulfuratus, and B. darkside. The advertisement calls of B. tridactylus is also distinct from that of B. albolineatus and B. mirissimus by having only isolates notes, instead of isolated notes and note groups.