Long Read Fragment Whole Genome Sequencing (lfrWGS)

Description

Current short-read based Whole Genome Sequencing (WGS) is the most widely used method for identifying genome-wide aberrations such as point mutations with read length typically less than several hundred base pairs. This short-read based WGS approach is proven to be highly informative in terms of detection of point mutations, indels and copy number alterations. However, short-read sequencing often results in sequences with scaffolding gaps, bias due to high GC content, repeat sequences and mis-aligned sequences. To interrogate the human genome at a higher resolution with better quality, the standard NGS workflow is challenged, especially for complex Structural Variation (SV) discovery, due to insufficient read coverage at breakpoints and loss of long-range genomic contiguity.

Technology

BGI’s innovative Single Tube Long Fragment Read (stLFR) technology produces long range information with accurate short-read sequencing, uniform coverage and superior SV detection capability, while maintaining reproducibility and consistency. With a small amount of input HMW genomic DNA (as low as 1 ng, approximately 300 genomic equivalents), added to a single tube containing 30 million barcoded beads, where the gDNA molecules are barcoded and subjected to random priming and polymerase amplification. Co-barcoded DNA fragments are then released followed by a modified library preparation process. The resulting libraries undergo DNA Nanoball (DNB™) generation and DNBSEQ sequencing. BGI’s proprietary computational algorithm uses the barcodes to assemble sequencing reads to the original HMW DNA molecule, enabling the construction of contiguous segments of phased variants.

Long Fragment Read WGS for Superior SV Detection

Compared with conventional WGS services, stLFR-based Long Fragment Read WGS (lfrWGS) delivers less bias, higher cover- age, higher reproducibility and near-complete genomic information. It significantly improves structural variants detection while maintaining the same excellent sensitivity for SNP, Indel and CNV detection. lfrWGS does not have the high error rate problem and high cost of long read sequencing platforms from Pacific Biosciences and Oxford Nanopore but provides a means to detect many of the complex genomic variants including SV by computationally maintaining the genomic contiguity.

Applications

• Germline Variant Detection.
• Somatic Variant Detection.
• Associating DNA Variants with Phenotype (Disease).
• Structural Variant Discovery (SV Discovery).
• Copy Number Variation Detection (CNV Detection).
• Biomarker Discovery.

Sequencing Quality Standard

• Co-barcoded LFR library preparation.
• PE100 DNBSEQ™ sequencing.
• Guaranteed ≥80% of bases with quality score of ≥Q30.
• CAP/CLIA laboratory services available.
• Standard and customized bioinformatics analysis available.
• Available data storage and bioinformatics applications.

Turn Around Time

Typical 40 working days from sample QC acceptance to filtered raw data availability.
Expedited services are available.

Sample Requirements

We can process your gDNA, whole blood, fresh frozen tissue and cell pellets, with the following general requirements:

• Genomic DNA > 500ng(c>1ng/ul).
• Whole blood > 1ml.
• Minimum DNA fragment size should be higher than 23kb.
• Shipment on dry ice.

FAQ

Q1. How does stLFR technology enable obtaining long-fragment information?

By using stLFR technology, each long gDNA molecule will be labeled with a unique barcode, resulting that all short fragments derived from the same long molecule share the same barcode. While barcodes between long molecules are different. After sequencing, both reads information and the corresponding barcode sequences will be obtained. As such, through barcode information, we can restore the reads information of long gDNA molecules.

Q2. How to store the long-fragment DNA?

After extraction, the long-fragment gDNA can be stored for half year at 4 °C and one year at - 20 °C. However, it is recommended to freeze and thaw only once at -20 °C. Repeated freeze[1]thaw cycles will cause sample degradation, and the sample DNA cannot be shaken or vigorously mixed to avoid long fragment DNA breakage.

Q3. What can stLFR technology do?

Combined with DNBSEQTM sequencing technology, stLFR technology enables high quality small variation detection, diploid genome phasing, large structural variation analysis, highly homologous genes mutation calling, etc., applying to areas such as reproductive health, cancer mechanisms, and single-gene diseases.

Q4. Is there any paper published using stLFR technology?

Yes, several articles have been published which shows that stLFR not only detects SNPs and InDels which conventional WGS can do, but also large structure variation (CNV, SV) on the genome through phasing and co-barcoded information. This mutation information can better explain the process of tumor metastasis. Notably, detection rate of the known SVs in NA12878 can be 100%.

Q5. What advantages of lfrWGS comparing with long-read and short-reads sequencing?

Accurate identification of DNA mutation using single-molecule long-read sequencing with a single nucleotide error rate of 5-15% is especially difficult. While short-read sequencing has a bias in detecting SV due to short inserts, which cannot be resolved by algorithm upgrade. Additionally, DNA input of stLFR could be as low as 1ng.