June 1, 2021  |  

HLA sequencing using SMRT Technology – High resolution and high throughput HLA genotyping in a clinical setting

Sequence based typing (SBT) is considered the gold standard method for HLA typing. Current SBT methods are rather laborious and are prone to phase ambiguity problems and genotyping uncertainties. As a result, the NGS community is rapidly seeking to remedy these challenges, to produce high resolution and high throughput HLA sequencing conducive to a clinical setting. Today, second generation NGS technologies are limited in their ability to yield full length HLA sequences required for adequate phasing and identification of novel alleles. Here we present the use of single molecule real time (SMRT) sequencing as a means of determining full length/long HLA sequences. Moreover we reveal the scalability of this method through multiplexing approches and determine HLA genotyping calls through the use of third party Gendx NGSengine® software.


June 1, 2021  |  

Evaluation of multiplexing strategies for HLA genotyping using PacBio Sequencing technology.

Fully phased allele-level sequencing of highly polymorphic HLA genes is greatly facilitated by SMRT Sequencing technology. In the present work, we have evaluated multiple DNA barcoding strategies for multiplexing several loci from multiple individuals, using three different tagging methods. Specifically MHC class I genes HLA-A, -B, and –C were indexed via DNA Barcodes by either tailed primers or barcoded SMRTbell adapters. Eight different 16-bp barcode sequences were used in symmetric & asymmetric pairing. Eight DNA barcoded adapters in symmetric pairing were independently ligated to a pool of HLA-A, -B and –C for eight different individuals, one at a time and pooled for sequencing on a single SMRT Cell. Amplicons generated from barcoded primers were pooled upfront for library generation. Eight symmetric barcoded primers were generated for HLA class I genes. These primers facilitated multiplexing of 8 samples and also allowed generation of unique asymmetric pairings for simultaneous amplification from 28 reference genomic DNA samples. The data generated from all 3 methods was analyzed using LAA protocol in SMRT analysis V2.3. Consensus sequences generated were typed using GenDx NGS engine HLA-typing software.


June 1, 2021  |  

Multiplexing human HLA class I & II genotyping with DNA barcode adapters for high throughput research.

Human MHC class I genes HLA-A, -B, -C, and class II genes HLA-DR, -DP and -DQ, play a critical role in the immune system as major factors responsible for organ transplant rejection. The have a direct or linkage-based association with several diseases, including cancer and autoimmune diseases, and are important targets for clinical and drug sensitivity research. HLA genes are also highly polymorphic and their diversity originates from exonic combinations as well as recombination events. A large number of new alleles are expected to be encountered if these genes are sequenced through the UTRs. Thus allele-level resolution is strongly preferred when sequencing HLA genes. Pacific Biosciences has developed a method to sequence the HLA genes in their entirety within the span of a single read taking advantage of long read lengths (average >10 kb) facilitated by SMRT technology. A highly accurate consensus sequence (=99.999 or QV50 demonstrated) is generated for each allele in a de novo fashion by our SMRT Analysis software. In the present work, we have combined this imputation-free, fully phased, allele-specific consensus sequence generation workflow and a newly developed DNA-barcode-tagged SMRTbell sample preparation approach to multiplex 96 individual samples for sequencing all of the HLA class I and II genes. Commercially available NGS-go reagents for full-length HLA class I and relevant exons of class II genes were amplified for hi-resolution HLA sequencing. The 96 samples included 72 that are part of UCLA reference panel and had pre-typing information available for 2 fields, based on gold standard SBT methods. SMRTbell adapters with 16 bp barcode tags were ligated to long amplicons in symmetric pairing. PacBio sequencing was highly effective in generating accurate, phased sequences of full-length alleles of HLA genes. In this work we demonstrate scalability of HLA sequencing using off the shelf assays for research applications to find biological significance in full-length sequencing.


June 1, 2021  |  

Full-length isoform sequencing of the human MCF-7 cell line using PacBio long reads.

While advances in RNA sequencing methods have accelerated our understanding of the human transcriptome, isoform discovery remains a challenge because short read lengths require complicated assembly algorithms to infer the contiguity of full-length transcripts. With PacBio’s long reads, one can now sequence full-length transcript isoforms up to 10 kb. The PacBio Iso- Seq protocol produces reads that originate from independent observations of single molecules, meaning no assembly is needed. Here, we sequenced the transcriptome of the human MCF-7 breast cancer cell line using the Clontech SMARTer® cDNA preparation kit and the PacBio RS II. Using PacBio Iso-Seq bioinformatics software, we obtained 55,770 unique, full-length, high-quality transcript sequences that were subsequently mapped back to the human genome with = 99% accuracy. In addition, we identified both known and novel fusion transcripts. To assess our results, we compared the predicted ORFs from the PacBio data against a published mass spectrometry dataset from the same cell line. 84% of the proteins identified with the Uniprot protein database were recovered by the PacBio predictions. Notably, 251 peptides solely matched to the PacBio generated ORFs and were entirely novel, including abundant cases of single amino acid polymorphisms, cassette exon splicing and potential alternative protein coding frames.


June 1, 2021  |  

Highly contiguous de novo human genome assembly and long-range haplotype phasing using SMRT Sequencing

The long reads, random error, and unbiased sampling of SMRT Sequencing enables high quality, de novo assembly of the human genome. PacBio long reads are capable of resolving genomic variations at all size scales, including SNPs, insertions, deletions, inversions, translocations, and repeat expansions, all of which are important in understanding the genetic basis for human disease and difficult to access via other technologies. In demonstration of this, we report a new high-quality, diploid aware de novo assembly of Craig Venter’s well-studied genome.


June 1, 2021  |  

The “Art” of shotgun sequencing

2015 SMRT Informatics Developers Conference Presentation Slides: Jason Chin of PacBio highlighted some of the challenges for shotgun assembly while suggesting some potential solutions to obtain diploid assemblies, including the FALCON method.


June 1, 2021  |  

Full-length sequencing of HLA class I genes of more than 1000 samples provides deep insights into sequence variability

Aim: The vast majority of donor typing relies on sequencing exons 2 and 3 of HLA class I genes (HLA-A, -B, -C). With such an approach certain allele combinations do not result in the anticipated “high resolution” (G-code) typing, due to the lack of exon-phasing information. To resolve ambiguous typing results for a haplotype frequency project, we established a whole gene sequencing approach for HLA class I, facilitating also an estimation of the degree of sequence variability outside the commonly sequenced exons. Methods: Primers were developed flanking the UTR regions resulting in similar amplicon lengths of 4.2-4.4 kb. Using a 4-primer approach, secondary primers containing barcodes were combined with the gene specific primers to obtain barcoded full-gene amplicons in a single amplification step. Amplicons were pooled, purified, and ligated to SMRT bells (i.e. annealing points for sequencing primers) following standard protocols from Pacific Biosciences. Taking advantage of the SMRT chemistry, pools of 48-72 amplicons were sequenced full length and phased in single runs on a Pacific Biosciences RSII instrument. Demultiplexing was achieved using the SMRT portal. Sequence analysis was performed using NGSengine software (GenDx). Results: We successfully performed full-length gene sequencing of 1003 samples, harboring ambiguous typings of either HLA-A (n=46), HLA-B (n=304) or HLA-C (n=653). Despite the high per-read raw error rates typical for SMRT sequencing (~15%) the consensus sequence proved highly reliable. All consensus sequences for exons 2 and 3 were in full accordance with their MiSeq-derived sequences. Unambiguous allelic resolution was achieved for all samples. We observed novel intronic, exonic as well as UTR sequence variations for many of the alleles covered by our data set. This included sequences of 600 individuals with HLA-C*07:01/C*07:02 genotype revealing the extent of sequence variation outside the exons 2 and 3. Conclusion: Here we present a whole gene amplification and sequencing approach for HLA class I genes. The maturity of this approach was demonstrated by sequencing more than 1000 samples, achieving fully phased allelic sequences. Extensive sequencing of one common allele combination hints at the yet to discover diversity of the HLA system outside the commonly analyzed exons.


June 1, 2021  |  

Phased full-length SMRT Sequencing of HLA DPB1

Aim: In contrast to exon-based HLA-typing approaches, whole gene genotyping crucially depends on full-length sequences submitted to the IMGT/HLA Database. Currently, full-length sequences are provided for only 7 out of 520 HLA-DPB1 alleles. Therefore, we developed a fully phased whole-gene sequencing approach for DPB1, to facilitate further exploration of the allelic structure at this locus. Methods: Primers were developed flanking the UTR-regions of DPB1 resulting in a 12 kb amplicon. Using a 4-primer approach, secondary primers containing barcodes were combined with the gene-specific primers to obtain barcoded full-gene amplicons in a single amplification step. Amplicons were pooled, purified, and ligated to SMRT bells (i.e. annealing points for sequencing primers) following standard protocols from Pacific Biosciences. Taking advantage of the SMRT chemistry, pools of 48 amplicons were sequenced full length in single runs on a Pacific Biosciences RSII instrument. Demultiplexing was performed using the SMRT portal. Sequence analysis was performed using the NGSengine software (GenDx). Results: We analyzed a set of 48 randomly picked samples. With 3 exceptions due to PCR failure, all genotype assignments conformed to standard genotyping results based on exons 2 and 3. Allelic proportions for heterozygous positions were evenly distributed (range 0.4 – 0.6) for all samples, suggesting unbiased amplifications. Despite the high per-read raw error rates typical for SMRT sequencing (~15%) the consensus sequence proved highly reliable. All consensus sequences for exons 2 and 3 were in full accordance with their MiSeq-derived sequences. We describe novel intronic sequence variation of the 7 so far genomically defined alleles, as well as 7 whole-length DPB1 alleles with hitherto unknown intronic regions. One of these alleles (HLA-DPB1*131:01) is classified as rare. Conclusion: Here we present a whole gene amplification and sequencing workflow for DPB1 alleles utilizing single molecule real-time (SMRT) sequencing from Pacific Biosciences. Validation of consensus sequences against known exonic sequences highlights the reliability of this technology. This workflow will facilitate amending the IMGT/HLA Database for DPB1.


June 1, 2021  |  

Access full spectrum of polymorphisms in HLA class I & II genes, without imputation for disease association and evolutionary research.

MHC class I and II genes are critically monitored by high-resolution sequencing for organ transplant decisions due to their role in GVHD. Their direct or linkage-based causal association, have increased their prominence as targets for drug sensitivity, autoimmune, cancer and infectious disease research. Monitoring HLA genes can however be tricky due to their highly polymorphic nature. Allele-level resolution is thus strongly preferred. However, most studies were historically focused on peptide binding domains of the HLA genes, due to technological challenges. As a result knowledge about the functional role of polymorphisms outside of exons 2 and 3 of HLA genes was rather limited. There are also relatively few full-length gene references currently available in the IMGT HLA database. This made it difficult to quickly adopt high-throughput reference-reliant methods for allele-level HLA sequencing. Increasing awareness regarding role of regulatory region polymorphisms of HLA genes in disease association1, nonetheless have brought about a revolution in full-length HLA gene sequencing. Researchers are now exploring ways to obtain complete information for HLA genes and integrate it with the current HLA database so it can be interpreted used by clinical researchers. We have explored advantages of SMRT Sequencing to obtain fully phased, allele-specific sequences of HLA class I and II genes for 96 samples using completely De novo consensus generation approach for imputation-free 4-field typing. With long read lengths (average >10 kb) and consensus accuracy exceeding 99.999% (Q50), a comprehensive snapshot of variants in exons, introns and UTRs could be obtained for spectrum of polymorphisms in phase across SNP-poor regions. Such information can provide invaluable insights in future causality association and population diversity research.


June 1, 2021  |  

Full-length cDNA sequencing of alternatively spliced isoforms provides insight into human cancer

The majority of human genes are alternatively spliced, making it possible for most genes to generate multiple proteins. The process of alternative splicing is highly regulated in a developmental-stage and tissue-specific manner. Perturbations in the regulation of these events can lead to disease in humans (1). Alternative splicing has been shown to play a role in human cancer, muscular dystrophy, Alzheimer’s, and many other diseases. Understanding these diseases requires knowing the full complement of mRNA isoforms. Microarrays and high-throughput cDNA sequencing have become highly successful tools for studying transcriptomes, however these technologies only provide small fragments of transcripts and building complete transcript isoforms has been very challenging (2). We have developed a technique, called Iso-Seq sequencing, that is capable of sequencing full-length, single-molecule cDNA sequences. The method employs SMRT Sequencing from PacBio, which can sequence individual molecules with read lengths that average more than 10 kb and can reach as long as 40 kb. As most transcripts are from 1 – 10 kb, we can sequence through entire RNA molecules, requiring no fragmentation or post-sequencing assembly. Jointly with the sequencing method, we developed a computational pipeline that polishes these full-length transcript sequences into high-quality, non-redundant transcript consensus sequences. Iso-Seq sequencing enables unambiguous identification of alternative splicing events, alternative transcriptional start and polyA sites, and transcripts from gene fusion events. Knowledge of the complete set of isoforms from a sample of interest is key for accurate quantification of isoform abundance when using any technology for transcriptome studies (3). Here we characterize the full-length transcriptome of paired tumor/normal samples from breast cancer using deep Iso-Seq sequencing. We highlight numerous discoveries of novel alternatively spliced isoforms, gene-fusion events, and previously unannotated genes that will improve our understanding of human cancer. (1) Faustino NA and Cooper TA. Genes and Development. 2003. 17: 419-437(2) Steijger T, et al. Nat Methods. 2013 Dec;10(12):1177-84.(3) Au KF, et al. Proc Natl Acad Sci U S A. 2013 Dec 10;110(50):E4821-30.


June 1, 2021  |  

Epigenome characterization of human genomes using the PacBio platform

In addition to the genome and transcriptome, epigenetic information is essential to understand biological processes and their regulation, and their misregulation underlying disease. Traditionally, epigenetic DNA modifications are detected using upfront sample preparation steps such as bisulfite conversion, followed by sequencing. Bisulfite sequencing has provided a wealth of knowledge about human epigenetics, however it does not access the entire genome due to limitations in read length and GC- bias of the sequencing technologies used. In contrast, Single Molecule, Real-Time (SMRT) DNA Sequencing is unique in that it can detect DNA base modifications as part of the sequencing process. It can thereby leverage the long read lengths and lack of GC bias for more comprehensive views of the human epigenome. I will highlight several examples of this capability towards the generation of new biological insights, including the resolution of methylation states in repetitive and GC-rich regions of the genome, and large-scale changes in the methylation status across a cancer genome as a function of drug sensitivity.


June 1, 2021  |  

HLA variant identification techniques

The Human Leukocyte Antigen (HLA) genes located on chromosome 6 are responsible for regulating immune function via antigen presentation and are one of the determining factors for stem cell and organ transplantation compatibility. Additionally various alleles within this region have been implicated in autoimmune disorders, cancer, vaccine response and both non-infectious and infectious disease risk. The HLA region is highly variable; containing repetitive regions; and co-dominantly expressed genes. This complicates short read mapping and means that assessing the effect of variation within a gene requires full phase information to resolve haplotypes.One solution to the problem of HLA identification is the use of statistical inference to suggest the most likely diploid alleles given the genotypes observed. The assumption of this approach is the availability of an extensive reference panel. Whilst there exists good population genetics data for imputing European populations, there remains a paucity of information about variation in African populations. Filling this gap is one of the aims of the Genome Diversity in Africa Project and as a first step we are performing a pilot study to identify the optimal method for determining HLA type information for large numbers of samples from African populations.To that end we have obtained samples from 125 consented African participants selected from 5 populations across Africa (Morrocan, Ashanti, Igbo, Kalenjin, and Zulu). The methods included in our pilot study are Sanger sequencing (ABI), NGS on HiSeqX Ten platform (Illumina); long-range PCR combined with single molecule real-time (SMRT) sequencing (PacBio); and for a subset of samples library preparation on GemCode Platform (10x Genomics), which delivers valuable long range contextual information, combined with Illumina NGS sequencing.Results from capillary sequencing suggests the presence of a minimum of two novel alleles. Long Range PCR have been performed initially on a subset of samples using both primers sourced from GenDX and designed as described in Shiina et al (2012). Initial results from both primer sets were promising on Promega DNA test samples but only the GenDX primers proved effective on the African samples, producing consistently PCR products of the expected size in the Igbo, Ashanti, Morrocan and Zulu samples. We will present early results from our evaluation of the different sequencing technologies


June 1, 2021  |  

Profiling metagenomic communities using circular consensus and Single Molecule, Real-Time Sequencing

There are many sequencing-based approaches to understanding complex metagenomic communities, spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR amplification. Whole-sample shotgun experiments require a high depth of coverage. As such, rare community members may not be represented in the resulting assembly. Circular-consensus, Single Molecule, Real-Time (SMRT) Sequencing reads in the 1-2 kb range, with >99% consensus accuracy, can be efficiently generated for low amounts of input DNA, e.g. as little as 10 ng of input DNA sequenced in 4 SMRT Cells can generate >100,000 such reads. While throughput is low compared to second-generation sequencing, the reads are a true random sampling of the underlying community. Long read lengths translate to a high number of the reads harboring full genes or even full operons for downstream analysis. Here we present the results of circular-consensus sequencing on a mock metagenomic community with an abundance range of multiple orders of magnitude, and compare the results with both 16S and shotgun assembly methods. We show that even with relatively low sequencing depth, the long-read, assembly-free, random sampling allows to elucidate meaningful information from the very low-abundance community members. For example, given the above low-input sequencing approach, a community member at 1/1,000 relative abundance would generate 100 1-2 kb sequence fragments having 99% consensus accuracy, with a high probability of containing a gene fragment useful for taxonomic classification or functional insight.


June 1, 2021  |  

SMRT Sequencing of the alala genome

Single Molecule Real-Time (SMRT) Sequencing was used to generate long reads for whole genome shotgun sequencing of the genome of the`alala (Hawaiian crow). The ‘alala is endemic to Hawaii, and the only surviving lineage of the crow family, Corvidae, in the Hawaiian Islands. The population declined to less than 20 individuals in the 1990s, and today this charismatic species is extinct in the wild. Currently existing in only two captive breeding facilities, reintroduction of the ‘alala is scheduled to begin in the Fall of 2016. Reintroduction efforts will be assisted by information from the ‘alala genome generated and assembled by SMRT Technology, which will allow detailed analysis of genes associated with immunity, behavior, and learning. Using SMRT Sequencing, we present here best practices for achieving long reads for whole genome shotgun sequencing for complex plant and animal genomes such as the ‘alala genome. With recent advances in SMRTbell library preparation, P6-C4 chemistry and 6-hour movies, the number of useable bases now exceeds 1 Gb per SMRT Cell. Read lengths averaging 10 – 15 kb can be routinely achieved, with the longest reads approaching 70 kb. Furthermore, > 25% of useable bases are in reads greater than 30 kb, advantageous for generating contiguous draft assemblies of contig N50 up to 5 Mb. De novo assemblies of large genomes are now more tractable using SMRT Sequencing as the standalone technology. We also present guidelines for planning out projects for the de novo assembly of large genomes.


June 1, 2021  |  

Full-length cDNA sequencing for genome annotation and analysis of alternative splicing

In higher eukaryotic organisms, the majority of multi-exon genes are alternatively spliced. Different mRNA isoforms from the same gene can produce proteins that have distinct properties and functions. Thus, the importance of understanding the full complement of transcript isoforms with potential phenotypic impact cannot be understated. While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants. The Iso-Seq protocol developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences to survey transcriptome isoform diversity useful for gene discovery and annotation. Knowledge of the complete isoform repertoire is also key for accurate quantification of isoform abundance. As most transcripts range from 1 – 10 kb, fully intact RNA molecules can be sequenced using SMRT Sequencing without requiring fragmentation or post-sequencing assembly. Our open-source computational pipeline delivers high-quality, non-redundant sequences for unambiguous identification of alternative splicing events, alternative transcriptional start sites, polyA tail, and gene fusion events. We applied the Iso-Seq method to the maize (Zea mays) inbred line B73. Full-length cDNAs from six diverse tissues were barcoded and sequenced across multiple size-fractionated SMRTbell libraries. A total of 111,151 unique transcripts were identified. More than half of these transcripts (57%) represented novel, sometimes tissue-specific, isoforms of known genes. In addition to the 2250 novel coding genes and 860 lncRNAs discovered, the Iso-Seq dataset corrected errors in existing gene models, highlighting the value of full-length transcripts for whole gene annotations.


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