Long-Read Sequencing

Using single-cell whole-genome long read sequencing results as an example. (Hård et al., 2023)

Reference

1. Hård J, Mold J E, Eisfeldt J, et al. Long-read whole-genome analysis of human single cells[J]. Nature Communications, 2023, 14(1): 5164.

1. What is the difference between Illumina sequencing and long-read sequencing?

Illumina sequencing, referred to as short-read sequencing, yields shorter sequence reads, typically between 100 and 300 base pairs long. Conversely, long-read sequencing technologies, like those offered by Oxford Nanopore Technologies and Pacific Biosciences, generate significantly longer reads, extending from thousands to tens of thousands of base pairs. This variance in read length enables long-read sequencing to enhance the resolution of intricate genomic regions, including repetitive sequences and structural variants, leading to a more thorough genomic understanding compared to Illumina sequencing. The application of long-read sequencing greatly assists in the deciphering of complex bacterial genomes, particularly when combined with short-read Illumina data through hybrid assembly methods.

2. What research fields is long-read sequencing suitable for?

Long-read sequencing is applicable to various research domains within genomics, including human genetics, evolutionary biology, microbiology, and botany. It enables researchers to gain a more comprehensive understanding of genome structure and function, thereby driving advancements in these fields.

3. What are the advantages and disadvantages of the PacBio platform?

The PacBio platform boasts high single-molecule accuracy, with over 90% of sequences achieving a quality score (Q) of above 30, and offers sequencing data with alignment accuracy surpassing that of NGS platforms. Moreover, it provides an average read length that surpasses NGS platforms by more than two orders of magnitude. However, its drawbacks include significantly higher sequencing costs compared to platforms like Novaseq, high equipment expenses, and the inability to interpret sequencing data in real-time, thus failing to meet the demands of scenarios requiring high timeliness.

4. What are the advantages and disadvantages of ONT technology?

The advantages of ONT technology lie in its high sequencing timeliness and relatively lower sequencing costs compared to the PacBio platform. Moreover, its average read length surpasses that of NGS platforms by more than two orders of magnitude. However, its disadvantages stem from systematic errors in signal detection, such as difficulties in identifying base homopolymers, resulting in significant differences in both single-molecule accuracy and alignment accuracy compared to PacBio and NGS platforms. These limitations restrict its applications.

The landscape of genomic structural variation in Indigenous Australians

Journal: Nature
Impact factor: 45.16
Published: 13 December 2023

Background

Australia hosts hundreds of Aboriginal nations, each with rich cultural and linguistic diversity. Despite extensive documentation of their cultural heritage, the genomic diversity of Indigenous Australians is underexplored, with significant under-representation in global genomic databases. The National Centre for Indigenous Genomics (NCIG) addresses this gap by engaging Aboriginal communities in ethical and community-led genomics research. In this study, population-scale long-read sequencing was conducted on Aboriginal communities using Oxford Nanopore Technologies, revealing previously uncharted genomic variations and structural variants, which are crucial for advancing genomic medicine.

Methods

Results

The complete T2T-chm13 genome reference was used to evaluate mappability and structural variant (SV) detection, showing ONT's superior alignment and SV detection. This revealed 159,912 unique SVs and 136,797 large indels, and identified 11 high-confidence copy number variants (CNVs) per individual, highlighting the benefits of long-read sequencing for detailed genomic profiling.

Fig. 1 Long-read sequencing in Indigenous Australian communities.

To characterize genomic structural variation, the authors categorized variants by type, size, and context. Most variants (84.9%) were repetitive sequences, including STRs, TRs, and mobile element insertions/deletions. CNVs were fewer but covered significant genome regions. Variants were unevenly distributed, with higher density near telomeres, particularly TR-associated SVs. Size varied by type, with TR-SVs larger than STRs and non-repetitive SVs. Mobile element SVs showed size peaks corresponding to major repeat families like Alu, L1, and SVA. Many SVs were unique to this study, especially given the inclusion of under-represented Australian communities and the use of long-read sequencing. Comparing this study's SVs to global databases, the authors found around 38% similarity with annotated SVs, indicating a high proportion of novel SVs.

Fig. 2 Landscape of genomic structural variation.

The authors analyzed genomic structural variations (SVs) among Indigenous and non-Indigenous Australians, finding most SVs to be private or polymorphic. Notably, 48.5% of SVs were unique to Indigenous individuals, while 9.2% were unique to non-Indigenous individuals. Unique SVs in Indigenous individuals were often unannotated and specific to particular communities. Genetic distinctions were evident, with distinct clusters for different communities in principal coordinate analysis, and shared SVs among Indigenous individuals were rare, underscoring their unique genomic diversity.

Fig. 3 Distribution of SVs in Indigenous and non-Indigenous individuals.

Fig. 4 Distribution of SVs between Indigenous communities.

Conclusion

This study unveils the structural variation landscape of Indigenous Australians using long-read sequencing, revealing diverse variations unique to this population. This underscores the need for ancestry-specific reference data in genomic medicine. We highlight the genetic heterogeneity among Indigenous communities and provide insights into broader patterns of genomic structural variation in human populations, particularly in short tandem repeats (STRs), which have implications for disease diagnosis and treatment.

Reference

1. Reis, A.L.M., Rapadas, M., Hammond, J.M. et al. The landscape of genomic structural variation in Indigenous Australians. Nature 624, 602–610 (2023).