Endoscopic ultrasound-guided fine-needle aspiration pancreatic adenocarcinoma samples yield adequate DNA for next-generation sequencing:A cohort analysis

BACKGROUND Genetic tests are increasingly performed for the management of unresectable pancreatic cancer.For genotyping aimed samples current guidelines recommend using core specimens,although based on moderate quality evidence.However,in clinical practice among the endoscopic ultrasound(EUS) guided tissue acquisition methods,fine needle aspiration(FNA) is the most widely performed.AIM To assess the adequacy for next generation sequencing(NGS) of the DNA yielded from EUS-FNA pancreatic adenocarcinoma(PDAC) samples.METHODS Between November 2018 and December 2021,105 patients with PDAC confirmed by EUS-FNA were included in the study at our tertiary gastroenterology center.Either 22 gauge(G) or 19G FNA needles were used.One pass was dedicated to DNA extraction.DNA concentration and purity(A260/280,A260/230) were assessed by spectrophotometry.We assessed the differences in DNA parameters according to needle size and tumor characteristics(size,location) and the adequacy of the extracted DNA for NGS(defined as A260/280 ≥ 1.7,and DNA yield:≥ 10 ng for amplicon based NGS,≥ 50 ng for whole exome sequencing [WES],≥ 100 ng for whole genome sequencing [WGS]) by analysis of variance and ttest respectively.Moreover,we compared DNA purity parameters across the different DNA yield categories.RESULTS Our cohort included 49% male patients,aged 67.02 ± 8.38 years.The 22G needle was used in 71%of the cases.The DNA parameters across our samples varied as follows:DNA yield:1289 ng(inter quartile range:534.75-3101),A260/280 = 1.85(1.79-1.86),A260/230 = 2.2(1.72-2.36).DNA yield was > 10 ng in all samples and > 100 ng in 93% of them(one sample < 50 ng).There were no significant differences in the concentration and A260/280 between samples by needle size.Needle size was the only independent predictor of A260/230 which was higher in the 22G samples(P =0.038).NGS adequacy rate was 90% for 19G samples regardless of NGS type,and for 22G samples it reached 89% for WGS adequacy and 91% for WES and amplicon based NGS.Samples with DNA yield > 100 ng had significantly higher A260/280(1.89 ± 0.32 vs 1.34 ± 0.42,P = 0.013).Tumor characteristics were not corelated with the DNA parameters.CONCLUSION EUS-FNA PDAC samples yield DNA adequate for subsequent NGS.DNA amount was similar between 22G and 19G FNA needles.DNA purity parameters may vary indirectly with needle size.

Sequencing of hepatitis C virus cDNA with polymerase chain reaction directed sequencing *

AIM To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome, and establish a new sequencing method in China. METHODS Polymerase Chain Reaction (PCR) was combined with DNA sequencing technique. PCR products were purified by agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), Polyethylene glycol (PEG) respectively. Then in the presence of a 5′ labeling PCR primer, purified PCR products were directly sequenced. By this method, HCV NS5b cDNA from two HCV infected individuals (HC 42 and HC 49) were sequenced.

DNA存储场景下的大小喷泉码模型设计

在DNA存储等应用场景中,传统喷泉码算法需要占用额外信道资源将源文件分组数目K传递给解码端。在实际应用中,虽然可以将K嵌入在每一个编码数据分组中进行传递,但这种做法会严重浪费信道的带宽。针对上述问题,提出了一种大小喷泉码模型,通过增加小喷泉码这一带外信道来优化关键参数的传递。小喷泉码将每个编码分组中有关参数K所占用空间的粒度降至1 bit,有效减少了带宽资源的消耗。此外,小喷泉码还能适应由于DNA存储介质不均匀所导致的编码序列不定长的限制条件,一定条件下甚至可以完全不占用额外信道带宽。

新型超声快速处理活检标本保存不同年限对DNA质量的影响

背景:新型超声组织处理技术越来越多地被用来进行分子生物学分析,研究新型超声处理不同存储年限组织DNA的质量,对进一步分子检测的标本质控具有重要意义。目的:探讨新型超声处理活检标本存储不同年限对DNA质量的影响,以期为分子检测探索最佳的标本存储时间。方法:收集40例乳腺穿刺小活检组织,采用超声技术制作石蜡标本,按照存储年限分为4组:<1年组、1-3年组、>3-5年组及>5年组,每组10例,对石蜡标本进行切片,每张切片厚3μm,切片10-15张,提取DNA后通过Nanophotometer N60超微量分光光度计和Qubit 4.0荧光计检测DNA的质量浓度,记录A_(260)/A_(280)比值判定DNA的纯度,利用全自动毛细管电泳核酸分析仪(Qsep 100)检测DNA片段完整性,以评估DNA片段的质量。结果与结论:4组样本A_(260)/A_(280)均值在1.8-2.0之间,达到纯度要求,无明显差异。4组样本的DNA质量浓度(Qubit浓度)均值分别为30.39,14.33,2.52,1.95 ng/μL;DNA的平均N/Q比值分别为6.48,14.18,24.56,29.86;DNA质量数均值分别为5.64,1.76,1.24,0.80;大片段占比均值分别为56.08%,17.72%,12.68%,7.90%。PCR检测内控基因Ct均值分别为15.32,17.09,18.39,21.24。与<1年组相比,其余3组DNA浓度显著降低,N/Q比值显著增加,DNA质量数和大片段占比均值显著降低,Ct值升高,差异有显著性意义(P<0.05)。实验结果表明,对于新型超声处理活检标本,应优先选择存储<1年的样本进行日常分子检测,储存3年内的样本可满足二代测序等检测要求,5年内样本仅可尝试进行PCR等检测,存储超过5年的样本不建议进行后续分子检测。

循环肿瘤DNA在膀胱癌中的研究进展及临床价值

随着生物技术的发展,液体活检技术在肿瘤方面的研究逐渐深入,循环肿瘤DNA(ctDNA)作为液体活检的主要方式之一,以其非侵入性、可重复性及可早期发现病变等特点备受关注。目前有研究表明ctDNA在膀胱癌(BCa)的早期诊断和分级、靶向治疗的选择和疗效分析、肿瘤的复发及进展等方面有一定的潜能,本文结合ctDNA的特点及其近年来在BCa中的研究进展和临床价值进行综述。ctDNA检测技术近年不断发展,人们在测序、突变测定技术发展及在增强其作为癌症检测生物标志物能力方面做出多项进展与尝试。对血浆样本中ctDNA的检测有助于在早期BCa的诊断中实现准确分类,但由于ctDNA含量低,需要更先进检测技术的支持;基于靶向测序的ctDNA分析是预测非肌层浸润性膀胱癌(NMIBC)患者疾病复发和侵袭性的一种有希望的方法。在晚期BCa患者中,可以应用ctDNA收集特有突变类型的特点,有针对性地指导用药、调整治疗及监测预后。尽管ctDNA在BCa的应用仍存在挑战,但已表现出广泛的应用前景。

DNA测序首段信号数据特别处理方法研究

针对荧光标记毛细管电泳DNA测序开始一段序列的信号杂乱特性以及不同碱基通道信号间的相对偏移问题,提出了一种用于首段信号特别处理的电泳迁移率自适应校正方法。分析设计基于四通道首段信号间峰重叠程度估计的运筹决策目标函数,通过目标函数动态寻优计算确定不同碱基通道信号的相对偏移系数,从而实现DNA测序首段信号的相对偏移校正,为提高首段信号碱基的合理判读及后续DNA精准排序提供了可靠的过程数据。

水生生物环境DNA监测技术的发展、应用与标准化

水生态系统是保障国家生态安全的重要基础。水生生物在水生态系统中扮演着核心角色,是研究水体演变的重要依据,是维护水生态健康的关键。传统水生生物调查和监测通常采用形态学方法,但其存在专业知识要求高、难以标准化和自动化及费时耗力等缺陷。环境DNA(Environmental DNA,简称eDNA)技术是一种通过监测环境中存在的DNA片段来识别特定生物物种的方法,可以实现基于水体中DNA分子进行水生生物的鉴定和监测,为水生生物的常态化监测提供了一个准确、便捷、可标准化和自动化实施的方案。文章介绍了eDNA技术的基本原理,总结回顾了eDNA技术从萌芽到广泛科研应用的发展历史和过程,介绍了基于eDNA的宏条形码和宏基因组等各类水生生物鉴定监测技术;阐述了eDNA技术在保护种、入侵种及生物类群监测和水生态评估等各领域的应用;分析了eDNA技术当前面临的物种参考序列数据库不完善等各类挑战;提出了通过优化完善数据库、样品采集方法、评价指标和参数、样品保藏、数据分析和存储等来推动eDNA技术标准化和自动化,以解决当前面临的挑战。同时,基于eDNA技术当前的发展阶段,提出了在我国水体结合专业形态分类鉴定开展水生生物eDNA技术标准化监测的实施建议。

DNA存储技术:挑战与未来

随着全球数据呈现指数级增长,当前的信息存储技术面临维护成本高昂、存储寿命有限等多个缺陷,逐渐无法满足日益凸显的需求。因此,迫切需要引入新的信息存储方法来解决这一问题。DNA作为一种天然的遗传信息载体,具备高存储密度、潜在低维护成本和长寿命等优势,因此被视为一种有潜力的新型信息存储介质。该文对DNA数据存储技术的基本原理和流程进行了概述,并回顾了其历史发展。同时,对当前基于DNA存储的领域仍面临的挑战进行了总结,如缓慢的数据写入和读取速度等,以及应对这些挑战的一些潜在策略。最后,为了满足全球对新存储方法的需求,该文指出了DNA数据存储技术的未来发展方向。

PCR-SSCP-DNA sequencing method in detecting PTEN gene mutation and its signifi cance in human gastric cancer

AIM: To discuss the possible effect of PTEN gene mutations on occurrence and development of gastric cancer. METHODS: Fifty-three gastric cancer specimens were selected to probe PTEN gene mutations in genome of gastric cancer and paracancerous tissues using PCR-SSCP-DNA sequencing method based on microdissection and to observe the protein expression by immunohistochemistry technique. RESULTS: PCR-SSCP-DNA sequencing indicated that 4 kinds of mutation sites were found in 5 of 53 gastric cancer specimens. One kind of mutation was found in exons. AA-TCC mutation was located at 40bp upstream of 3’ lateral exon 7 (115946 AA-TCC). Such mutations led to terminator formation in the 297th codon of the PTEN gene. The other 3 kinds of mutation were found in introns,including a G-C point mutation at 91 bp upstream of 5’ lateral exon 5(90896 G-C),a T-G point mutation at 24 bp upstream of 5’ lateral exon 5 (90963 T-G),and a single base A mutation at 7 bp upstream of 5’ lateral exon 5 (90980 A del). The PTEN protein expression in gastric cancer and paracancerous tissues detected using immunohistochemistry technique indicated that the total positive rate of PTEN protein expression was 66% in gastric cancer tissue,which was significantly lower than that (100%) in paracancerous tissues (P < 0.005). CONCLUSION: PTEN gene mutation and expression may play an important role in the occurrence and development of gastric cancer.

重叠延伸PCR生成长重复DNA序列及其在纳米孔测序中的应用

短链DNA的纳米孔测序存在孔道利用率低、数据质量差等问题。因此,亟需建立一种用纳米孔测序技术准确测定短链DNA序列的方法。本研究以1 nmol/L完全互补的序列为引物,以短链DNA为模板,在72℃退火温度下进行PCR,实现了短链DNA的重叠延伸,最终生成长串联重复序列,其产物长度为原始长度的5~20倍。生成的长链DNA可直接测序,仅需对齐单个或少数几个Reads进行简单分析即可获得精确的序列信息。本研究所开发的重叠延伸法将纳米孔One read精确度提高至99.28%,这对提高纳米孔测序的精度具有重要作用。

A stage-scanning laser confocal microscope and protocol for DNA methylation sequencing

Recent understanding of the role of epigenetic regulation in health and disease has necessitated the development of newer and efficient methods to map the methylation pattern of target gene. In this article we report construction of a stage-scanning laser confocal microscope (SLCM) and associated protocol that determines the methylation status of target gene. We have adapted restricted Sanger’s sequencing where fluorescine labeled primers and dideoxy guanine fraction alone are used for target amplification and termination at cytosine positions. Amplified ssDNA bands are separated in 6% denaturing PAGE and scanned using SLCM to sequence the positions of methylated cytosines. We demonstrate that our me- thodology can detect < 100 femtomoles of DNA, and resolve the position of cytosine within ± 2 nucleotide. In a calibration run using a designer DNA of 99 bases, our methodology had resolved all the 11 cytosine positions of the DNA. We have further demonstrated the utility of apparatus by mapping methylation status in the Exon-1 region of a gene, E-Cadherin, in the plasma DNA sample of a healthy subject. We believe our approach constitute a low cost alternative to conventional DNA sequencers and can help develop methylation based DNA biomarkers for the diagnosis of disease and in therapeutics.

Distinctive Drug-resistant Mutation Profiles and Interpretations of HIV-1 Proviral DNA Revealed by Deep Sequencing in Reverse Transcriptase

Objective To investigate distinctive features in drug-resistant mutations （DRMs） and interpretations for reverse transcriptase inhibitors （RTIs） between proviral DNA and paired viral RNA in HIV-l-infected patients. Methods Forty-three HIV-l-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA. Results Compared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M1841 and M2301 were more prevalent in proviral DNA than in viral RNA （Fisher＇s exact test, P〈0.05）. Considering ＇majority resistant variants＇, 15 samples （19.48%） showed differences in drug resistance interpretation between viral RNA and proviral DNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering ＇minority resistant variants＇, 22 samples （28.57%） were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA. Conclusion Compared with viral RNA, the distinctive information of DRMs and drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.

不同单细胞全基因组扩增体系扩增牛微量血液DNA效果评价

旨在通过二代测序技术评估不同单细胞全基因组扩增(single cell whole genome amplification,scWGA)体系对牛微量血液基因组DNA的扩增效果,建立微量DNA全基因组扩增体系。本研究分别采用MDA(multiple displacement amplification)和MALBAC(multiple annealing and looping-based amplification cycles)两种scWGA体系对华西牛1 ng血液基因组DNA进行全基因组扩增,随后基于两种体系的扩增产物以及原始未稀释血液DNA构建测序文库,利用DNBSEQ-T7RS测序平台进行全基因组测序(whole genome sequencing,WGS),通过比较扩增产物片段大小、浓度、总质量评估扩增效率,通过分析GC含量、测序覆盖度、基因分型一致率、基因型检出率等评估两种体系的扩增效果。结果显示,MDA体系的扩增产物片段大于MALBAC体系(8 kb vs.0.2~2 kb),产物浓度和总质量显著高于MALBAC体系(P<0.05)。基于测序数据,1×和5×测序深度下,MDA体系的基因组覆盖度显著高于MALBAC体系(P<0.05),此外,MDA体系的分型一致率、检出率显著高于MALBAC体系,而等位基因缺失率、假阳性率显著低于MALBAC体系(P<0.05)。综上,本研究揭示了MDA和MALBAC两种扩增体系基于华西牛1 ng血液基因组DNA扩增的体系特点,为改进现有华西牛胚胎基因组选择中的关键扩增技术提供理论依据,促进华西牛遗传育种进展。

Complementary DNA sequencing (cDNA): an eff ective approach for assessing the diversity and distribution of marine benthic ciliates along hydrographic gradients

The Yellow Sea Cold Water Mass(YSCWM)is a distinct hydrographic phenomenon of the Yellow Sea,and the distribution pattern of meio-and macrobenthos diff ers inside and outside of the YSCWM.However,such a pattern has never been observed in the microbenthic ciliate communities.Therefore,we hypothesized that benthic ciliates followed a similar distribution pattern as meio-and macrobenthos,but this pattern has not been uncovered by morphological methods.We evaluated the diversity and distribution of benthic ciliates at fi ve stations along hydrographic gradients across the YSCWM and adjacent shallow water by using morphology and DNA and complementary DNA(cDNA)high-throughput sequencing of the V4 region of 18S rRNA gene.Results showed that the diversity of benthic ciliates detected by DNA(303 OTUs),and the cDNA(611 OTUs)sequencing was much higher than that detected by the morphological method(79 species).Morphological method detected roughly diff erent ciliate communities inside and outside of the YSCWM,but without statistical signifi cance.No clear pattern was obtained by DNA sequencing.In contrast,cDNA sequencing revealed a distinct distribution pattern of benthic ciliate communities like meioand macrobenthos,which coincided well with the results of the environmental parameter analysis.More than half of the total sequences detected by DNA sequencing belonged to planktonic ciliates,most(if not all)of which were recovered from historic DNA originating through the sedimentation of pelagic forms because none of them were observed morphologically.The irrelevant historic DNA greatly infl uenced the recovery of rare species and thus limited the understanding of the benthic ciliate diversity and distribution.Our research indicates that the methods used have signifi cant eff ects on the investigation of benthic ciliate communities and highlights that cDNA sequencing has great advantages in estimating the diversity and distribution of benthic ciliates,as well as the potential for benthic environmental assessments.

Species Identification by Means of Mitochondrial Cytochrome <i>b</i>DNA Sequencing in Processed Anchovy, Sardine and Tuna Products

Identifying the contents of processed food products is essential to correct labelling. In processed foodstuffs, species identification through morphological analysis is difficult. Several factors hinder the identification of fish species in processed foods: proteins or other materials subjected to analysis may be denatured during heat treatments;the presence of other ingredients (e.g., olive and other vegetable oils) may interfere with the analysis. Consequently, possible frauds perpetrated by replacing valuable species with less precious ones may go undetected. In most processed samples (e.g. canned products), DNA is degraded into small fragments, which considerably reduces the sensitivity of molecular analysis. The main goal of our research was to develop an analytical method able to identify fish species in highly processed products, such as canned fish. The assay was developed by combining an effective method of DNA recovery from samples with the detection of small-sized sequences of the mitochondrial Cytb gene. This method appears particularly suitable when morphological characterization is difficult, to carry out such as in canned products where DNA is degraded or present in small quantities. We have analyzed 60 samples of seafood commercial products identifying 3 different genera and five different species. All analyzed samples revealed a correct species declaration, for one sample we highlighted important commercial fraud. We also used bio-informatic identification systems for the Sequence Alignment and the construction of phylogenetic tree to better confirm the revealed fraud.

Genome Sequencing Using Graph Theory Approach

Genome sequencing is the process of determining in which order the nitrogenous bases also known as nucleotides within a DNA molecule are arranged. Every organism’s genome consists of a unique sequence of nucleotides. These nucleotides bases provide the phenotypes and genotypes of a cell. In mathematics, Graph theory is the study of mathematical objects known as graphs which are made of vertices (or nodes) connected by either directed edges or indirect edges. Determining the sequence in which these nucleotides are bonded can help scientists and researchers to compare DNA between organisms, which can help show how the organisms are related. In this research, we study how graph theory plays a vital part in genome sequencing and different types of graphs used during DNA sequencing. We are going to propose several ways graph theory is used to sequence the genome. We are as well, going to explore how the graphs like Hamiltonian graph, Euler graph, and de Bruijn graphs are used to sequence the genome and advantages and disadvantages associated with each graph.

DNA Sequencing Modified Method through Effective Regulation of Its Translocation Speed in Aqueous Solution

Solid-state nanopore DNA sequencing modified method is developed. Method is based on the tunnel current investigation through the nanogap made on lateral gold electrodes in the form of nanowires or nanoribbons. The movement of DNA in aqueous solution is regulated by the potential applied to reference electrode. The potential applied to the lateral metal electrodes helps to the creation of the molecular junctions. They consist of the nucleosides passing through the pores. Taking into account that DNA moves under gravity, electrophoretic and drag forces, the analytic expression for the DNA translocation speed is calculated and analyzed. The conditions for decreasing the DNA translocation speed or increasing the nucleosides reading time are received. It is shown that one can control value of the DNA molecules bases reading time and the frequency of the bases passes by the choice of magnitude of the potential applied to reference electrode. Our results, therefore potentially suggest a realistic, inherently design-specific, high-throughput nanopore DNA sequencing device/cell as a de-novo alternative to the existing methods.

基于SCAR标记和DNA条形码技术的苍术基原鉴别研究

目的开发出能同时鉴别北苍术和关苍术的分子标记方法,并探究不同种质资源苍术的遗传进化关系。方法对不同地区北苍术Atractylodes chinensis(Bunge)Koidz及关苍术A.japonica Koidz.ex Kitam基因组DNA的差异片段进行测序,结合SRAP、ISSR、DAMD分子标记方法,优化PCR反应体系,筛选并转换成特异性标记,同时,采用条形码方法分析种间序列差异。结果通过SRAP、ISSR、DAMD三种分子标记方法的PCR扩增,共筛选出198对能稳定扩增且重现性好的引物,转换出7对能稳定、快速鉴别北苍术和关苍术的SCAR引物。条形码方法检测出北苍术ITS2序列长度为454 bp,关苍术ITS2序列长度为453 bp,与其他苍术属植物之间遗传距离较远。NJ树结果显示,北苍术、关苍术及其他苍术属植物均各自聚为一支,表现出良好的单系性。依据ITS2二级结构,4种苍术属植物在螺旋区的茎环数目、大小、位置均有明显差异,可以直观地进行区分。结论所开发的特异性SCAR标记为苍术属植物优良品种的筛选提供了新方法,DNA条形码能稳定、准确鉴别北苍术。

Automatic DNA sequencing for electrophoresis gels using image processing algorithms

DNA electrophoresis gel is an important biologically experimental technique and DNA sequencing can be defined by it. Traditionally, it is time consuming for biologists to exam the gel images by their eyes and often has human errors during the process. Therefore, automatic analysis of the gel image could provide more information that is usually ignored by human expert. However, basic tasks such as the identification of lanes in a gel image, easily done by human experts, emerge as problems that may be difficult to be executed automatically. In this paper, we design an automatic procedure to analyze DNA gel images using various image processing algorithms. Firstly, we employ an enhanced fuzzy c-means algorithm to extract the useful information from DNA gel images and exclude the undesired background. Then, Gaussian function is utilized to estimate the location of each lane of A, T, C, and G on the gels images automatically. Finally, the location of each band on the gel image can be detected accurately by tracing lanes, renewing lost bands, and eliminating repetitive bands.

DNA Sequencing: Current State and Prospects of Development

In this review, we collected and classified the stages of development of DNA sequencing methods and described its peculiarities. We pay attention mostly on solid-stead nanopore sequencing methods. Detailed discussion of the peculiarity and feasibility of the electrical methods of DNA sequencing is discussed. The detail analyses of the literature data, some critical considerations and the potential ways of optimization of DNA nanopore sequencing were presented.