Science unlocked: publication picks from March 2024

Human genomics

1. Gapless assembly of complete human and plant chromosomes using only nanopore sequencing (bioRxiv)

This study investigates the viability of Oxford Nanopore sequencing as a standalone method for 'telomere-to-telomere' genome assembly, challenging the necessity of multi-platform approaches. The focus is on the accuracy and contiguity of sequencing data, using the human genome HG002 research sample and plant samples.

Key points:

• Achieved highly accurate duplex reads with median Q-scores above 30, demonstrating comparability to reads from an alternative long-read technology in Q-scores but with better consistency across different read lengths
• Duplex + ultra-long nanopore reads provided exceptional contiguity (NG50: 103 Mb), surpassing the NG50 of 101.76 for a previously published hybrid assembly combining ultra-long nanopore reads and reads from an alternative long-read technology
• Generated consensus Q-scores for the human genome of 55.77 with the Oxford Nanopore sequencing method alone, marking the only high-quality gapless assembly from a single platform to date
• Showed fewer missing genes than the previously published hybrid assembly, with more T2T contigs
• ‘When combined with the affordable yet high throughput Oxford Nanopore P2 sequencer, the single-instrument, T2T assembly recipes presented here open the exciting possibility of personalized human genomes and complete genomes for any other species, in any country and potentially any institution in the world.'

Figure 1 Comparable QV-scores were identified for both nanopore duplex sequencing data and data from an alternative long-read technology for chromosome X of HG002 (~QV:30), yet duplex showed consistent quality across various read lengths, while HiFi's longer reads had reduced quality due to fewer subread passes, impacting consensus accuracy.

Explore duplex sequencing and PromethION 2

2. Nanopore sequencing of 1000 Genomes Project samples to build a comprehensive catalogue of human genetic variation (medRxiv)

The 1000 Genomes Project ONT Sequencing Consortium (1KGP-ONT) is dedicated to leveraging long nanopore reads to produce high-coverage, high-quality data for over 800 samples. This preprint reports findings from the first 100 samples sequenced on R9.4.1 Flow Cells, with the remaining samples to be sequenced on the latest R10.4.1 technology (watch this space).

Key points:

• Demonstrated higher recall and precision for SNVs with Oxford Nanopore versus short-read sequencing, particularly in well-characterised genomic areas, along with promising indel identification capabilities
• Highlighted the resolution of complex alleles in pharmacogenes and repeat expansions associated with diseases, such as the detailed resolution of CYP2D6 variations, crucial for potential pharmacogenomic applications and personalised medicine (see figure)
• In contrast, equivalent short-read data struggled to resolve this complex variation, highlighting the potential clinical significance of long nanopore reads for accurate genetic characterisation and its potential to improve treatment efficacy by enabling personalised medication

Figure 3. Phased IGV view of Oxford Nanopore data data displays a CYP2D6 full-gene deletion in haplotype 1 and a hybrid tandem insertion (*36+*10) in haplotype 2 of HG02396, contrasting with the complexity obscured in short-read whole genome sequencing, where the reads could not be phased.

Epigenetics

3. A comparison of methods for detecting DNA methylation from long-read sequencing of human genomes (Genome Biology)

Researchers from deCODE genetics performed an extensive evaluation of CpG methylation detection tools for Oxford Nanopore sequencing using a large cohort of 7,179 DNA samples. This study included a comparison with both oxidative bisulfite sequencing and sequencing via an alternative long-read technology, emphasising the advancements in nanopore technology.

Key points:

• Observed a reduction in strand bias when utilizing the newer Oxford Nanopore R10.4.1 Flow Cells compared to the older R9.4.1
• Enhanced detection of CpGs in difficult-to-read genomic areas was attributed to better read alignments from long-read sequencing methods
• Nanopore sequencing detected the highest number of CpG sites, outperforming the results from both oxidative bisulfite sequencing and the alternative long-read platform
• Confirmed the high accuracy and consistency of CpG methylation detection in nanopore-sequenced samples in comparison to oxidative bisulfite sequencing

Plant Genomics

4. A nanopore-based cucumber genome assembly reveals structural variations at two QTLs controlling hypocotyl elongation (Plant Physiology)

This research presents a significant advancement in cucumber genomics by using long nanopore sequencing reads to achieve a remarkably contiguous and complete assembly. With an improved BUSCO score of 97.9%, the study showcases the depth of sequencing achieved.

Key points:

• The XIS49 cucumber genome assembly reached 314.23 Mb, substantially expanding the known genetic landscape compared to the previous 224.80 Mb reference
• Demonstrated a significant increase in read length N50 to ~35 kb, a notable improvement over the ~10 kb read length N50 of the prior reference generated using an alternative long-read technology
• Enabled more effective variant calling and the detection of structural variants (SVs), with about 10,000 SVs identified
• Identified critical genetic alterations, such as a 4 kb deletion in the PhyB gene associated with stem length and adaptability to low light, highlighting the assembly's potential for detailed genetic studies

Discover more applications of nanopore technology in plant research.

Microbiology & Infectious Diseases

5. Latest RNA and DNA nanopore sequencing allows for rapid avian influenza profiling (bioRxiv)

This preprint underscores the importance of tracking avian influenza virus (AIV) distribution and mutations. It evaluates the efficacy of nanopore sequencing, comparing a direct cDNA approach to the direct RNA sequencing (the latest Direct RNA Sequencing Kit, SQK-RNA004, and the previous iteration, SQK-RNA002) for profiling AIV.

Key points:

• Found that the new Direct RNA Sequencing Kit, SQK-RNA004, provided significantly better coverage than the previous iteration, SQK-RNA002
• SQK-RNA004 achieved accuracy on par with the high-accuracy DNA-based R10 nanopore sequencing
• Successfully identified m6A modifications using the latest direct RNA sequencing chemistry, particularly within the M segment, demonstrating a major step forward in direct RNA sequencing for viral studies

Figure 2. SQK-RNA004 showing much improved throughput compared to the predecessor SQK-RNA002 kit, with huge gains observed at the 3’ end.

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