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【资讯翻译】Detection of Genomic Rearrangements Using Capture Based NGS

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发表于 2016-3-17 10:36:40 | 显示全部楼层 |阅读模式
Detection of Genomic Rearrangements Using Capture Based NGS

Introduction: Genomic rearrangements in inherited disease and cancer involve gross alterations including deletions, duplications, insertions, inversions or translocations. Unbiased capture based NGS analysis has been proven to be an effective approach to simultaneously detect single nucleotide variations (SNVs) and copy number variations (CNVs) due to genomic rearrangements. In this study, we demonstrated the detection of Alu insertions, exonic deletions and duplications by capture based NGS.

Method: we developed an analytical workflow to detect potential genomic rearrangements involving coding exons and adjacent introns. After removal of the low quality reads, the reads with ≥80% match sequences were re-aligned to a reference sequence, and nucleotide changes with ≥5% variant calls were scored.

Results: Assessment of the NGS data using the filtering parameters described above identified below genomic rearrangements: 1) Alu insertion and other novel large insertion: homozygous Alu insertion in the MAK gene in 3 unrelated patients with retinitis pigmentosa (patient 1-3); heterozygous Alu insertion in the PYGL gene in two unrelated patients with clinical indication suggestive of glycogen storage disease (patient 4-5); a heterozygous novel insertion of  279bp in MUT gene in a patient with biochemical diagnosis of mut(0) methylmalonic aciduria (patient 6). The insAlu in MAK gene is a founder mutation in Jewish population. Two heterozygous pathogenic variants, c.1874_1875insAlu and c.243+2T>C in the PYGL gene, were detected in GSD patient 4. The c.1874_1875insAlu is a novel insertion of Alu element in exon 16 of the PYGL gene. Two heterozygous variants, c.501_502insAlu and c.1822G>A (p.G608S) in the PYGL gene, were detected in GSD patient 5. The c.501_502insAlu is an insertion in exon 4 of the PYGL gene. A heterozygous c.372_374dupGGA(p.Lys124_Asp125insGlu) variant and a heterozygous novel insertion c.146_147ins279 in the exon 1 of the MUT gene were detected in patient 6. PCR and Sanger sequencing confirmed that the 279bp insertion sequence is from 3’UTR of the ENO1 gene. Subsequent RT-PCR analysis confirmed these two changes are in trans configuration. 2) exonic deletions: multiple exons deletion in the BRCA1 gene, exons 10_14del and exons 13_16del, were detected in two unrelated females with breast cancer and family history of cancer (patient 7-8). 3) Copy number gain: an exon 11 duplication and a heterozygous c.3350G>A (p.R1117K) variant in the PALB2 gene were detected in a woman with breast cancer (patient 9). This exon 11 duplication was confirmed to be tandem by PCR/sequencing across the junction. In a patient with indication of visual impairment, a heterozygous c.1654C>T (p.R552W) variant and a copy number gain of exons 4 through 10 of the PDE6B gene were detected (patient 10).


Conclusions:  Two main mechanisms may apply for above described rearrangements: direct insertions of Alu elements or other sequence within coding region and disrupt normal reading frame, and unequal homologous recombination events between Alu repeats or other low copy repeats. The single end NGS reads is only about 100bp, which is impossible to capture the full length of insertions ~300bp, or other genomic rearrangements. Using a loose filter setting to retain misaligned reads is critical to capture junction sequence. Re-alignment of the misaligned reads can lead to the mapping the breakpoints and the origin of the inserted sequence. Our results demonstrate that capture based NGS test is a powerful tool to detect genomic rearrangements when the junction points reside in the targeted regions. The ability to identify Alu insertions and other genomic rearrangements dramatically increases the clinical utility of capture NGS approach and underscores the importance of maximizing the utility of the capture NGS data by resetting the analytical parameters to reveal additional sequence information.   

信源地址:https://acmg.expoplanner.com/ind ... amp;session_id=1511

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发表于 2016-3-18 22:12:12 | 显示全部楼层
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发表于 2016-3-19 01:05:36 | 显示全部楼层
基于高通量测序技术(NGS)捕获基因组重排

       简介:在遗传疾病和癌症中,基因组重排产生恶劣的变化,包括缺失、重复、插入或易位,倒置。基于NGS分析的无差别捕获技术已被证明是能够同时检测单核苷酸变异的(SNV)和由基因组重排导致的拷贝数变异(CNVs)的有效方法。在这项研究中,我们进行了基于NGS捕获的Alu插入、外显子缺失和重复检测
       方法:我们开发了一个分析工作流程,以检测潜在的包含编码外显子和相邻的内含子在内的基因组重排。在去除低质量阅读后,序列匹配≥80%的阅读重新与参考序列进行比对,并对≥5%以上核苷酸发生突变的阅读进行评分。
       结果:使用上述滤波参数对NGS数据进行分析,并确定了如下的基因组重排的:1)Alu序列和其他新的大片段插入:在3例视网膜色素变性的不相关病例中(1-3号患者)发现MAK基因中Alu插入的纯合突变;2例临床指征提示糖原贮积病的不相关病理(4-5号患者)中发现PYGL基因中Alu插入的杂合突变;1例生化诊断出mut(0)甲基丙二酸尿症病人中发现MUT基因中插入279bp新序列的杂合突变(6号患者)。MAK基因中的insAlu是犹太人口的创始者突变。4号糖原贮积病患者中检测到两个分别在PYGL基因c.1874_1875insalu和 c.243+2T>C上的杂合致病突变。c.1874_1875insalu是一种新型的在PYGL基因第16号外显子Alu元件插入。5号患者中检测到两例分别在PYGL基因c.501_502insalu和c.1822g >(p.g608s)位的杂合突变。c.501_502insalu是在 PYGL基因4号外显子的插入。6号患者中检测到c.372_374dupGGA(p.lys124_asp125insglu)的杂合突变和MUT基因c.146_147ins279的新型插入的杂合突变。PCR法和Sanger测序证实插入的279bp序列来自ENO1基因的3'UTR。随后的RT-PCR分析证实了这两个变化是反式构型。2)外显子缺失:在两名不相关患有乳腺癌和癌症家族史的女性(7-8患者)中检测到BRCA1基因的多个外显子缺失, 10_14号外显子缺失和 13_16号外显子缺失。3)拷贝数增加:在一例患有乳腺癌的中检测到PALB2基因的11号外显子重复和c.3350G>A(p.R1117K)的杂合突变(9号患者)。交界处的PCR/测序证实外显子11重复为串联。在一例具有视觉障碍的适应症的病人中检测到PDE6B基因的c.1654c>T(p.r552w)杂合突变和4至10号外显子拷贝数增加(10号患者)。
       结论:上述重排可以归因于两种主要机制:在编码区直接插入Alu元件或其他序列,从而扰乱正常的阅读框;Alu重复或其他低拷贝重复之间的不平等同源重组事件。由于NGS单末端阅读取仅约100bp,所以难以捕捉插入或其他基因组重排的全长序列(~ 300bp)。使用一个松散的过滤设置以保留错误比对阅读对于捕获连接区序列是非常关键的。对错误比对阅读的重新比对可以获得插入序列断点和起始位置的图谱。我们的研究结果表明,当连接点位于目标区域时,基于NGS的捕获测试是一个检测基因组重排的强大工具。基于NGS的捕获测试能够识别Alu插入和其他基因组重排,这极大地提高了其临床实用性。需要强调的是,通过重置分析参数显示其他序列信息对于使其数据得到最大化的利用具有重要意义。

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 楼主| 发表于 2016-3-21 08:53:05 | 显示全部楼层
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