Long range PCR-based deep sequencing for haplotype determination in mixed HCMV infections

Background Short read sequencing has been used extensively to decipher the genome diversity of human cytomegalovirus (HCMV) strains, but falls short to reveal individual genomes in mixed HCMV strain populations. Novel third-generation sequencing platforms offer an extended read length and promise to resolve how distant polymorphic sites along individual genomes are linked. In the present study, we established a long amplicon PacBio sequencing workflow to identify the absolute and relative quantities of unique HCMV haplotypes spanning over multiple hypervariable sites in mixtures. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-culture enriched viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections. Results Total substitution and indel error rate of mapped reads ranged from 0.17 to 0.43% depending on the stringency of quality trimming. Artificial HCMV DNA mixtures were correctly determined down to 1% abundance of the minor DNA source when the total HCMV DNA input was 4 × 104 copies/ml. PCR products of up to 7.7 kb and a GC content < 55% were efficiently generated when DNA was directly isolated from patient samples. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Alignments of distinct haplotype sequences within patient samples showed uneven distribution of sequence diversity, interspersed by long identical stretches. Moreover, diversity estimation at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed haplotype populations. Conclusions Quantitative haplotype determination by long amplicon sequencing provides a novel approach for HCMV strain characterisation in mixed infected samples which can be scaled up to cover the majority of the genome by multi-amplicon panels. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.

GREPore-seq: A Robust Workflow to Detect Changes after Gene Editing through Long-range PCR and Nanopore Sequencing

To achieve the enormous potential of gene-editing technology in clinical therapies, both the on-target and unintended editing consequences need to be thoroughly evaluated. However, there is a lack of a comprehensive, pipelined, large-scale and economical workflow for detecting genome editing outcomes, in particular insertion or deletion of a large fragment. Here, we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR-Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR products. To overcome the high error rates and indels of nanopore sequencing, we developed a pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing (GREPore-seq). GREPore-seq can detect NHEJ-mediated double-stranded oligodeoxynucleotide (dsODN) insertions with comparable accuracy to Illumina next-generation sequencing (NGS). GREPore-seq also identifies HDR-mediated large gene knock-in, which excellently correlates with FACS analysis data. Low-level plasmid backbone insertion after HDR editing was also detected. We have established a practical workflow to identify genetic changes, including quantifying dsODN insertions, knock-ins, plasmid backbone insertions, and large fragment deletions after CRISPR editing. This toolkit for nanopore sequencing of pooled long amplicons should have broad applications in assessing on-target HDR editing and inadvertent large indels of over 1 kb. GREPore-seq is freely available at GitHub (https://github.com/lisiang/GREPore-seq).

Extension of Mitogenome Enrichment Based on Single Long-Range PCR: mtDNAs and Putative Mitochondrial-Derived Peptides of Five Rodent Hibernators

Enriching mitochondrial DNA (mtDNA) for sequencing entire mitochondrial genomes (mitogenomes) can be achieved by single long-range PCR. This avoids interference from the omnipresent nuclear mtDNA sequences (NUMTs). The approach is currently restricted to the use of samples collected from humans and ray-finned fishes. Here, we extended the use of single long-range PCR by introducing back-to-back oligonucleotides that target a sequence of extraordinary homology across vertebrates. The assay was applied to five hibernating rodents, namely alpine marmot, Arctic and European ground squirrels, and common and garden dormice, four of which have not been fully sequenced before. Analysis of the novel mitogenomes focussed on the prediction of mitochondrial-derived peptides (MDPs) providing another level of information encoded by mtDNA. The comparison of MOTS-c, SHLP4 and SHLP6 sequences across vertebrate species identified segments of high homology that argue for future experimentation. In addition, we evaluated four candidate polymorphisms replacing an amino acid in mitochondrially encoded subunits of the oxidative phosphorylation (OXPHOS) system that were reported in relation to cold-adaptation. No obvious pattern was found for the diverse sets of mammalian species that either apply daily or multiday torpor or otherwise cope with cold. In summary, our single long-range PCR assay applying a pair of back-to-back primers that target a consensus sequence motif of Vertebrata has potential to amplify (intact) mitochondrial rings present in templates from a taxonomically diverse range of vertebrates. It could be promising for studying novel mitogenomes, mitotypes of a population and mitochondrial heteroplasmy in a sensitive, straightforward and flexible manner.

Long range PCR reveals the genetic cargo of IncP-1 plasmids in the complex microbial community of an on-farm biopurification system treating pesticide contaminated wastewater

Promiscuous plasmids like IncP-1 plasmids play an important role in the bacterial adaptation to pollution by acquiring and distributing xenobiotic catabolic genes. However, most information comes from isolates and the role of plasmids in governing community-wide bacterial adaptation to xenobiotics and other adaptive forces is not fully understood. Current information on the contribution of IncP-1 plasmids in community adaptation is limited because methods are lacking that directly isolate and identify the plasmid borne adaptive functions in whole-community DNA. In this study, we optimized long range PCR to directly access and identify the cargo carried by IncP-1 plasmids in environmental DNA. The DNA between the IncP-1 backbone genes trbP and traC , a main insertion site of adaptive trait determinants, is amplified and its content analysed by high-throughput sequencing. The method was applied to DNA of an on-farm biopurification system (BPS), treating pesticide contaminated wastewater, to examine whether horizontal gene exchange of catabolic functions by IncP-1 plasmids is a main driver of community adaptation in BPS. The cargo recovered from BPS community DNA, encoded catabolic but also resistance traits and various other (un)known functions. Unexpectedly, catabolic traits composed only a minor fraction of the cargo, indicating that the IncP-1 region between trbP and traC is not a major contributor to catabolic adaptation of the BPS microbiome. Instead, it contains a functionally diverse set of genes which either may assist biodegradation functions, be remnants of random gene recruitment, or confer other crucial functions for proliferation in the BPS environment. IMPORTANCE This study presents a long range PCR for direct and cultivation-independent access to the identity of the cargo of a major insertion hot spot of adaptive genes in IncP-1 plasmids and hence a new mobilome tool for understanding the role of IncP-1 plasmids in complex communities. The method was applied to DNA of an on-farm biopurification system (BPS) treating pesticide-contaminated wastewater, aiming at new insights on whether horizontal exchange of catabolic functions by IncP-1 plasmids is a main driver of community adaptation in BPS. Unexpectedly, catabolic functions represented a small fraction of the cargo genes while multiple other gene functions were recovered. These results show that the cargo of the target insertion hot spot in IncP-1 plasmids in a community, not necessarily relates to the main selective trait imposed on that community. Instead these functions might contribute to adaptation to unknown selective forces or represent remnants of random gene recruitment.

Three patients with defects in interferon gamma receptor signaling: a challenging diagnosis

Background: Defects in IFN–gamma receptor (IFN-γR) signaling via STAT1 leads to susceptibility to infection by otherwise weak pathogenic mycobacteria, resulting in mendelian susceptibility to mycobacterial disease. We identified three patients presented with disseminated mycobacterial infections caused by M. avium, M. persicum or M. bovis BCG respectively. Whole-exome sequencing (WES) was used as the first line diagnostic approach, however in all patients additional analysis was crucial to make the definite diagnosis. Method: WES, SNP array and long range PCR were performed to identify the genetic defects. Expression of IFNGR1, STAT1, CD64, SOCS1 and phosphorylation of STAT1 were determined after stimulation with IFN-α or IFN-γ. Results: In Patient 1, only one heterozygous variant p.(Val63Gly) in the IFNGR1 gene was identified by WES. Additional genetic analysis identified a second complex Alu-insertion in IFNGR1. Patient 2 was compound heterozygous for the null p.(Val68Lysfs*6) variant and the hypomorphic p.(Ile37Thr) variant in IFNGR1. In Patient 3 a novel variant in the STAT1 gene p.(Asn460Ile) was identified. Patients 1 and 2 had reduced expression of IFN-γR1. All patients had reduced phosphorylation of STAT1 and absent induction of SOCS1 after IFN-γ stimulation. While STAT1 phosphorylation was normal after IFN–α stimulation in Patient 1 and 2, and mildly reduced in Patient 3. Conclusion: We conclude that functional assays are crucial to assess the extent of IFN-γR signaling defects when new combinations of bi-allelic or non-conclusive genetic variants are found, which is important in the determination of clinical treatment.

Case Report: Prenatal Diagnosis for a Rett Syndrome Family Caused by a Novel MECP2 Deletion With Heteroduplexes of PCR Product

Rett syndrome is an X-linked dominant, postnatal neurological disorder. Approximately 80–90% of classic Rett syndrome patients harbor mutations in the coding region of MECP2. Somatic or germline MECP2 mosaicism is not rare, and paternal germline MECP2 mosaicism occurs in especially high proportions. Here, we report the case of a Chinese girl with Rett syndrome in whom a heterozygous deletion was found in exon 4 of MECP2 using multiplex ligation-dependent probe amplification. To obtain an accurate region of deletion, we narrowed down the deletion region using real-time quantitative PCR, and subsequent long-range PCR was performed to detect the deletion breakpoints. Surprisingly, three DNA bands from long-range PCR products were observed after gel electrophoresis. To exclude somatic mosaicism, we performed T-A cloning and DNA sequencing, the middle DNA band was proved to be a heteroduplex of the PCR product in vitro. Meanwhile, a prenatal diagnosis was performed for the pregnant mother of the patient. Our study showed that the patient was heterozygous for the deletion of 713-base pairs in exon 4 of MECP2 (MECP2: c.441_1153del713), resulting in a frameshift and premature termination of the 487 amino acid protein at the 154th codon. In summary, we reported a novel heterozygous deletion in the MECP2 gene with heteroduplexes of the PCR product in vitro, which can help in the genetic counseling and prenatal diagnosis of disorders of MECP2 defects.

Long Range PCR-based deep sequencing for haplotype determination in mixed HCMV infections

Background Short read sequencing, which has extensively been used to decipher the genome diversity of human cytomegalovirus (HCMV) strains, often falls short to assess co-linearity of non-adjacent polymorphic sites in mixed HCMV populations. In the present study, we established a long amplicon sequencing workflow to identify number and relative quantities of unique HCMV haplotypes in mixtures. Accordingly, long read PacBio sequencing was applied to amplicons spanning over multiple polymorphic sites. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-free viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections. Results Our data show that artificial HCMV DNA mixtures were correctly determined upon long amplicon sequencing down to 1% abundance of the minor DNA source. Total error rate of mapped reads ranged from 0.17 to 0.43 depending on the stringency of quality trimming. PCR products of up to 7.7 kb and a GC content < 55% were efficiently generated when DNA was directly isolated from bronchoalveolar lavage samples, yet long range PCR may display a slightly lower sensitivity compared to short amplicons. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Intra-patient haplotype diversity is unevenly distributed across the target site and often interspersed by long identical stretches, thus unable to be linked by short reads. Moreover, diversity at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed populations. Conclusions Quantitative haplotype determination by long amplicon sequencing provides a novel approach for HCMV strain characterisation in mixed infected samples which can be scaled up to cover the majority of the genome. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.

New Approach for Detection of Normal Alternative Splicing Events and Aberrant Spliceogenic Transcripts with Long-Range PCR and Deep RNA Sequencing

RNA sequencing is a promising technique for detecting normal and aberrant RNA isoforms. Here, we present a new single-gene, straightforward 1-day hands-on protocol for detection of splicing alterations with deep RNA sequencing from blood. We have validated our method’s accuracy by detecting previously published normal splicing isoforms of STK11 gene. Additionally, the same technique was used to provide the first comprehensive catalogue of naturally occurring alternative splicing events of the NBN gene in blood. Furthermore, we demonstrate that our approach can be used for detection of splicing impairment caused by genetic variants. Therefore, we were able to reclassify three variants of uncertain significance: NBN:c.584G>A, STK11:c.863-5_863-3delCTC and STK11:c.615G>A. Due to the simplicity of our approach, it can be incorporated into any molecular diagnostics laboratory for determination of variant’s impact on splicing.

Long Range PCR-based deep sequencing for haplotype determination in mixed HCMV infections

Short read sequencing, which has extensively been used to decipher the genome diversity of human cytomegalovirus (HCMV) strains, often falls short to assess co-linearity of non-adjacent polymorphic sites in mixed HCMV populations. In the present study, we established a long amplicon sequencing workflow to identify number and relative quantities of unique HCMV haplotypes in mixtures. Accordingly, long read PacBio sequencing was applied to amplicons spanning over multiple polymorphic sites. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-free viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections. Our data show that artificial HCMV DNA mixtures were correctly determined upon long amplicon sequencing down to 1% abundance of the minor DNA source. Total error rate of mapped reads ranged from 0.17 to 0.43 depending on the stringency of quality trimming. PCR products of up to 7.7 kb and a GC content <55% were efficiently generated when DNA was directly isolated from bronchoalveolar lavage samples, yet long range PCR may display a slightly lower sensitivity compared to short amplicons. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Intra-patient haplotype diversity is unevenly distributed across the target site and often interspersed by long identical stretches, thus unable to be linked by short reads. Moreover, diversity at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed populations. Quantitative haplotype determination by long amplicon sequencing provides a novel approach for HCMV strain characterisation in mixed infected samples which can be scaled up to cover the majority of the genome. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.

Accurate Molecular Diagnosis of Gaucher Disease Using Clinical Exome Sequencing as a First-Tier Test

Gaucher disease (GD) is an autosomal recessive lysosomal disorder due to beta-glucosidase gene (GBA) mutations. The molecular diagnosis of GD is complicated by the presence of recombinant alleles originating from a highly homologous pseudogene. Clinical exome sequencing (CES) is a rapid genetic approach for identifying disease-causing mutations. However, copy number variation and recombination events are poorly detected, and further investigations are required to avoid mis-genotyping. The aim of this work was to set-up an integrated strategy for GD patients genotyping using CES as a first-line test. Eight patients diagnosed with GD were analyzed by CES. Five patients were fully genotyped, while three were revealed to be homozygous for mutations that were not confirmed in the parents. Therefore, MLPA (multiplex ligation-dependent probe amplification) and specific long-range PCR were performed, and two recombinant alleles, one of them novel, and one large deletion were identified. Furthermore, an MLPA assay performed in one family resulted in the identification of an additional novel mutation (p.M124V) in a relative, in trans with the known p.N409S mutation. In conclusion, even though CES has become extensively used in clinical practice, our study emphasizes the importance of a comprehensive molecular strategy to provide proper GBA genotyping and genetic counseling.