Sequence Fusion Algorithm of Tumor Gene Sequencing and Alignment Based on Machine Learning

With the rapid development of DNA high-throughput testing technology, there is a high correlation between DNA sequence variation and human diseases, and detecting whether there is variation in DNA sequence has become a hot research topic at present. DNA sequence variation is relatively rare, and the establishment of DNA sequence sparse matrix, which can quickly detect and reason fusion variation point, has become an important work of tumor gene testing. Because there are differences between the current comparison software and mutation detection software in detecting the same sample, there are errors between the results of derivative sequence comparison and the detection of mutation. In this paper, SNP and InDel detection methods based on machine learning and sparse matrix detection are proposed, and VarScan 2, Genome Analysis Toolkit (GATK), BCFtools, and FreeBayes are compared. In the research of SNP and InDel detection with intelligent reasoning, the experimental results show that the detection accuracy and recall rate are better when the depth is increasing. The reasoning fusion method proposed in this paper has certain advantages in comparison effect and discovery in SNP and InDel and has good effect on swelling and pain gene detection.

Optimization of Enzymatic Fragmentation is Crucial To Maximize Genome Coverage: A Comparison of Library Preparation Methods for Illumina Sequencing

Background Novel commercial kits for whole genome library preparation for next-generation sequencing on Illumina platforms promise shorter workflows, lower inputs and cost savings. Time savings are achieved by employing enzymatic DNA fragmentation and by combining end-repair and tailing reactions. Fewer cleanup steps also allow greater DNA input flexibility (1 ng-1 µg), PCR-free options from 100 ng DNA, and lower price as compared to the well-established sonication and tagmentation-based DNA library preparation kits. Results We compared the performance of four enzymatic fragmentation-based DNA library preparation kits (from New England Biolabs, Roche, Swift Biosciences and Quantabio) to a tagmentation-based kit (Illumina) using low input DNA amounts (10 ng) and PCR-free reactions with 100 ng DNA. With four technical replicates of each input amount and kit, we compared the kits` fragmentation sequence-bias as well as performance parameters such as sequence coverage and the clinically relevant detection of single nucleotide and indel variants. While all kits produced high quality sequence data and demonstrated similar performance, several enzymatic fragmentation methods produced library insert sizes which deviated from those intended. Libraries with longer insert lengths performed better in terms of coverage, SNV and indel detection. Lower performance of shorter-insert libraries could be explained by loss of sequence coverage to overlapping paired-end reads, exacerbated by the preferential sequencing of shorter fragments on Illumina sequencers. We also observed that libraries prepared with minimal or no PCR performed best with regard to indel detection. Conclusions The enzymatic fragmentation-based DNA library preparation kits from NEB, Roche, Swift and Quantabio are good alternatives to the tagmentation based Nextera DNA flex kit from Illumina, offering reproducible results using flexible DNA inputs, quick workflows and lower prices. Libraries with insert DNA fragments longer than the cumulative sum of both read lengths avoid read overlap, thus produce more informative data that leads to strongly improved genome coverage and consequently also increased sensitivity and precision of SNP and indel detection. In order to best utilize such enzymatic fragmentation reagents, researchers should be prepared to invest time to optimize fragmentation conditions for their particular samples.

Ultraspecific somatic SNV and indel detection in single neurons using primary template-directed amplification

Primary template-directed amplification (PTA) is an improved amplification technique for single-cell DNA sequencing. We generated whole-genome analysis of 76 single neurons and developed SCAN2, a computational method to accurately identify both clonal and non-clonal somatic (i.e., limited to a single neuron) single nucleotide variants (SNVs) and small insertions and deletions (indels) using PTA data. Our analysis confirms an increase in non-clonal somatic mutation in single neurons with age, but revises estimates for the rate of this accumulation to be 15 SNVs per year. We also identify artifacts in other amplification methods. Most importantly, we show that somatic indels also increase by at least 2 indels per year per neuron and that indels may have a larger impact on gene function than somatic SNVs in human neurons.

Characterizing sensitivity and coverage of clinical WGS as a diagnostic test for genetic disorders

Background Due to its reduced cost and incomparable advantages, WGS is likely to lead to changes in clinical diagnosis of rare and undiagnosed diseases. However, the sensitivity and breadth of coverage of clinical WGS as a diagnostic test for genetic disorders has not been fully evaluated. Methods Here, the performance of WGS in NA12878, the YH cell line, and the Chinese trios were measured by assessing their sensitivity, PPV, depth and breadth of coverage using MGISEQ-2000. We also compared the performance of WES and WGS using NA12878. The sensitivity and PPV were tested using the family-based trio design for the Chinese trios. We further developed a systematic WGS pipeline for the analysis of 8 clinical cases. Results In general, the sensitivity and PPV for SNV/indel detection increased with mean depth and reached a plateau at an ~ 40X mean depth using down-sampling samples of NA12878. With a mean depth of 40X, the sensitivity of homozygous and heterozygous SNPs of NA12878 was > 99.25% and > 99.50%, respectively, and the PPV was 99.97% and 98.96%. Homozygous and heterozygous indels showed lower sensitivity and PPV. The sensitivity and PPV were still not 100% even with a mean depth of ~ 150X. We also observed a substantial variation in the sensitivity of CNV detection across different tools, especially in CNVs with a size less than 1 kb. In general, the breadth of coverage for disease-associated genes and CNVs increased with mean depth. The sensitivity and coverage of WGS (~ 40X) was better than WES (~ 120X). Among the Chinese trios with an ~ 40X mean depth, the sensitivity among offspring was > 99.48% and > 96.36% for SNP and indel detection, and the PPVs were 99.86% and 97.93%. All 12 previously validated variants in the 8 clinical cases were successfully detected using our WGS pipeline. Conclusions The current standard of a mean depth of 40X may be sufficient for SNV/indel detection and identification of most CNVs. It would be advisable for clinical scientists to determine the range of sensitivity and PPV for different classes of variants for a particular WGS pipeline, which would be useful when interpreting and delivering clinical reports.

CRISPR/Cas9-mediated knockout of clinically relevant alloantigenes in human primary T cells

Background The ability of CRISPR/Cas9 to mutate any desired genomic locus is being increasingly explored in the emerging area of cancer immunotherapy. In this respect, current efforts are mostly focused on the use of autologous (i.e. patient-derived) T cells. The autologous approach, however, has drawbacks in terms of manufacturing time, cost, feasibility and scalability that can affect therapeutic outcome or wider clinical application. The use of allogeneic T cells from healthy donors may overcome these limitations. For this strategy to work, the endogenous T cell receptor (TCR) needs to be knocked out in order to reduce off-tumor, graft-versus-host-disease (GvHD). Furthermore, CD52 may be knocked out in the donor T cells, since this leaves them resistant to the commonly used anti-CD52 monoclonal antibody lymphodepletion regimen aiming to suppress rejection of the infused T cells by the recipient. Despite the great prospect, genetic manipulation of human T cells remains challenging, in particular how to deliver the engineering reagents: virus-mediated delivery entails the inherent risk of altering cancer gene expression by the genomically integrated CRISPR/Cas9. This is avoided by delivery of CRISPR/Cas9 as ribonucleoproteins, which, however, are fragile and technically demanding to produce. Electroporation of CRISPR/Cas9 expression plasmids would bypass the above issues, as this approach is simple, the reagents are robust and easily produced and delivery is transient. Results Here, we tested knockout of either TCR or CD52 in human primary T cells, using electroporation of CRISPR/Cas9 plasmids. After validating the CRISPR/Cas9 constructs in human 293 T cells by Tracking of Indels by Decomposition (TIDE) and Indel Detection by Amplicon Analysis (IDAA) on-target genomic analysis, we evaluated their efficacy in primary T cells. Four days after electroporation with the constructs, genomic analysis revealed a knockout rate of 12–14% for the two genes, which translated into 7–8% of cells showing complete loss of surface expression of TCR and CD52 proteins, as determined by flow cytometry analysis. Conclusion Our results demonstrate that genomic knockout by electroporation of plasmids encoding CRISPR/Cas9 is technically feasible in human primary T cells, albeit at low efficiency.

A fusion method based on alignment software with SNP and Indel detection methods

Background: With the advent of the second generation sequencing technology, the discovery of sequence alignment and sequence variation is a long-standing challenge. Results: A method based on general alignment software, SNP and Indel software tools was proposed in this paper. By comparing the advantages of traditional alignment software, we can produce the best alignment. SNP and Indel detection tools fusion research found that different depth of sequencing effect is different. When the sequence depth reaches a certain value, select one of the software for testing. Conclusions: Finally, the experimental verification shows that SNP and Indel have certain advantages in the comparison of the effects of the fusion method.

INDEL detection, the ‘Achilles heel’ of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels

Advances in genome editing technologies have enabled manipulation of genomes at the single base level. These technologies are based on programmable nucleases (PNs) that include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) nucleases and have given researchers the ability to delete, insert or replace genomic DNA in cells, tissues and whole organisms. The great flexibility in re-designing the genomic target specificity of PNs has vastly expanded the scope of gene editing applications in life science, and shows great promise for development of the next generation gene therapies. PN technologies share the principle of inducing a DNA double-strand break (DSB) at a user-specified site in the genome, followed by cellular repair of the induced DSB. PN-elicited DSBs are mainly repaired by the non-homologous end joining (NHEJ) and the microhomology-mediated end joining (MMEJ) pathways, which can elicit a variety of small insertion or deletion (indel) mutations. If indels are elicited in a protein coding sequence and shift the reading frame, targeted gene knock out (KO) can readily be achieved using either of the available PNs. Despite the ease by which gene inactivation in principle can be achieved, in practice, successful KO is not only determined by the efficiency of NHEJ and MMEJ repair; it also depends on the design and properties of the PN utilized, delivery format chosen, the preferred indel repair outcomes at the targeted site, the chromatin state of the target site and the relative activities of the repair pathways in the edited cells. These variables preclude accurate prediction of the nature and frequency of PN induced indels. A key step of any gene KO experiment therefore becomes the detection, characterization and quantification of the indel(s) induced at the targeted genomic site in cells, tissues or whole organisms. In this survey, we briefly review naturally occurring indels and their detection. Next, we review the methods that have been developed for detection of PN-induced indels. We briefly outline the experimental steps and describe the pros and cons of the various methods to help users decide a suitable method for their editing application. We highlight recent advances that enable accurate and sensitive quantification of indel events in cells regardless of their genome complexity, turning a complex pool of different indel events into informative indel profiles. Finally, we review what has been learned about PN-elicited indel formation through the use of the new methods and how this insight is helping to further advance the genome editing field.

Characterizing sensitivity and coverage of clinical WGS as a diagnostic test for genetic disorders

Background: Due to its reduced cost and incomparable advantages, WGS is likely to lead to changes in clinical diagnosis of rare and undiagnosed diseases. However, the sensitivity and breadth of coverage of clinical WGS as a diagnostic test for genetic disorders has not been fully evaluated. Here, the performance of WGS in NA12878, the YH cell line, and the Chinese trios were measured by assessing their sensitivity, PPV, depth and breadth of coverage using MGISEQ-2000. We also compared the performance of WES and WGS using NA12878. The sensitivity and PPV were tested using the family-based trio design for the Chinese trios. We further developed a systematic WGS pipeline for the analysis of 8 clinical cases. Results: In general, the sensitivity and PPV for SNV/indel detection increased with mean depth and reached a plateau at an ~40X mean depth using down-sampling samples of NA12878. With a mean depth of 40X, the sensitivity of homozygous and heterozygous SNPs of NA12878 was >99.25% and >99.50%, respectively, and the PPV was 99.97% and 98.96%. Homozygous and heterozygous indels showed lower sensitivity and PPV. The sensitivity and PPV were still not 100% even with a mean depth of ~150X. We also observed a substantial variation in the sensitivity of CNV detection across different tools, especially in CNVs with a size less than 1 kb. In general, the breadth of coverage for disease-associated genes and CNVs increased with mean depth. The sensitivity and coverage of WGS (~40X) was better than WES (~120X). Among the Chinese trios with an ~40X mean depth, the sensitivity among offspring was >99.48% and >96.36% for SNP and indel detection, and the PPVs were 99.86% and 97.93%. All 12 previously validated variants in the 8 clinical cases were successfully detected using our WGS pipeline.Conclusions: The current standard of a mean depth of 40X may be sufficient for SNV/indel detection and identification of most CNVs. It would be advisable for clinical scientists to determine the range of sensitivity and PPV for different classes of variants for a particular WGS pipeline, which would be useful when interpreting and delivering clinical reports.