Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA Genes.
Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA Genes.
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DOI：
10.3389/fgene.2021.635601
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发表时间：
2021
影响因子：
3.7
通讯作者：
Saavalainen P
中科院分区：
文献类型：
--
作者：
Johansson T;Koskela S;Yohannes DA;Partanen J;Saavalainen P
由于人类白细胞抗原(人类白细胞抗原)基因的高度多态和嵌合性，从下一代测序(NGS)数据中识别人类白细胞抗原(HLA)等位基因具有挑战性。由于人类白细胞抗原基因的复杂性和等位基因分配的困难，牛津纳米孔技术公司(ONT)的单分子测序技术因其适用于长阅读测序而引起了人们的极大兴趣。除了读取长度，ONT的优势还在于它的便携性和快速实时测序的可能性，这使得能够同时进行数据分析。在这里，我们描述了一种使用ONT测序和SeqNext-HLA SeqPilot软件(JSI Medical Systems GmbH)的基于RNA的靶向HLA分型方法。12个经典的人类白细胞抗原基因从50个个体的DNA中浓缩，在10个Minion R9.4 SpotOn Flow细胞中进行条形码编码、汇集和测序，每个样本产生超过30,000个读数。使用条形码2D读取，SeqPilot将HLA等位基因分配给平均读取深度为1750倍的两场或更高的分型分辨率。序列分析在低分辨率水平(单场)下的准确率为99-100%，在高分辨率水平(两场)下的准确率为74-100%，与预期的等位基因一致。ONT RNA测序仍然存在一些局限性，如阅读噪声、均聚体错误以及缺乏可靠的算法，这些都干扰了确定的等位基因分配。这些问题需要在未来仔细检查，以提高等位基因呼叫率。然而，我们在这里表明，对ONT minion上的多重cDNA扩增子文库进行测序可以得到准确的高分辨率分型结果，对12个经典的HLA基因座进行分型。对于人类白细胞抗原的研究，ONT RNA测序是一种很有前景的方法，因为它能够对全长的人类白细胞抗原转录本进行测序。该技术除可用于人类白细胞抗原基因分型外，还可同时用于基因表达分析。
Identification of human leukocyte antigen (HLA) alleles from next-generation sequencing (NGS) data is challenging because of the high polymorphism and mosaic nature of HLA genes. Owing to the complex nature of HLA genes and consequent challenges in allele assignment, Oxford Nanopore Technologies’ (ONT) single-molecule sequencing technology has been of great interest due to its fitness for sequencing long reads. In addition to the read length, ONT’s advantages are its portability and possibility for a rapid real-time sequencing, which enables a simultaneous data analysis. Here, we describe a targeted RNA-based method for HLA typing using ONT sequencing and SeqNext-HLA SeqPilot software (JSI Medical Systems GmbH). Twelve classical HLA genes were enriched from cDNA of 50 individuals, barcoded, pooled, and sequenced in 10 MinION R9.4 SpotON flow cell runs producing over 30,000 reads per sample. Using barcoded 2D reads, SeqPilot assigned HLA alleles to two-field typing resolution or higher with the average read depth of 1750x. Sequence analysis resulted in 99–100% accuracy at low-resolution level (one-field) and in 74–100% accuracy at high-resolution level (two-field) with the expected alleles. There are still some limitations with ONT RNA sequencing, such as noisy reads, homopolymer errors, and the lack of robust algorithms, which interfere with confident allele assignment. These issues need to be inspected carefully in the future to improve the allele call rates. Nevertheless, here we show that sequencing of multiplexed cDNA amplicon libraries on ONT MinION can produce accurate high-resolution typing results of 12 classical HLA loci. For HLA research, ONT RNA sequencing is a promising method due to its capability to sequence full-length HLA transcripts. In addition to HLA genotyping, the technique could also be applied for simultaneous expression analysis.
影响因子：
5
作者：
Sillé FC;Conde L;Zhang J;Akers NK;Sanchez S;Maltbaek J;Riby JE;Smith MT;Skibola CF
通讯作者：
Skibola CF