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适用说明：本文档适用于货号为 KK2600(07958919001), KK2601(07958927001)及 KK2602 (07958935001)产品。

适用说明：本文档适用于KAPA独立双端标记接头试剂盒（08861919702）、KAPA独立双端标记接头板（08861862001）和单独的KAPA接头稀释缓冲液（08278539001）。

适用说明：本说明书适用于货号为KK8520 的KAPA Hyper Prep Kit for SureSelectXT Target Enrichment。
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RNA-Seq is a powerful method for transcriptome analysis used in varied field of biology. Although several commercial products and hand-made protocols enable us to prepare RNA-Seq library from total RNA, their cost are still expensive. Here, we established a low-cost and multiplexable whole mRNA-Seq library preparation method for illumine sequencers. In order to reduce cost, we used cost-effective and robust commercial regents with small reaction volumes. This method is a whole mRNA-Seq, which can be applied even to non-model organisms lacking the transcriptome references. In addition, we designed large number of 3′ PCR primer including 8 nucleotides barcode sequences for multiplexing up to three hundreds samples. To summarize, it is possible with this protocol to prepare 96 directional RNA-Seq libraries from purified total RNA in three days and can be pooled for up to three hundred libraries. This is beneficial for large scale transcriptome analysis in many fields of animals and plant biology.

适用说明：本文档适用于Illumina平台的KAPA文库定量试剂盒（07960166001、07960140001、07960204001、07960255001、07960336001和07960298001）、Illumina平台的KAPA文库定量引物和PCR混合物试剂盒 （07960441001、07960484001、07960522001、07960727001和07960573001）、Illumina平台的KAPA文库定量标准品和引物试剂盒（07960085001）、Illumina平台的KAPA文库定量标准品试剂盒（07960409001和07960409001）、Illumina平台的KAPA文库定量稀释对照试剂盒（07960417001）和Illumina平台的KAPA文库定量引物试剂盒（07960093001）。
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Detecting heteroplasmies in the mitochondrial DNA (mtDNA) has been a challenge for many years. In the past, Sanger sequencing was the main option to perform this analysis, however, this method could not detect low frequency heteroplasmies. Massive Parallel Sequencing (MPS) provides the opportunity to study the mtDNA in depth, but a controlled pipeline is necessary to reliably retrieve and quantify the low frequency variants. It has been shown that differences in methods can significantly affect the number and frequency of the retrieved variants.

In this protocol, we present a method involving both wet lab and bioinformatics that allows identifying and quantifying single nucleotide variants in the full mtDNA sequence, down to a heteroplasmic load of 1.5%. For this, we set up a PCR-based amplification of the mtDNA, followed by MPS using Illumina chemistry, and variant calling with two different algorithms, mtDNA server and Mutect.

The PCR amplification is used to enrich the mitochondrial fraction, while the bioinformatic processing with two algorithms is used to discriminate the true heteroplasmies from background noise. The protocol described here allows for deep sequencing of the mitochondrial DNA in bulk DNA samples as well as single cells (both large cells such as human oocytes, and small-sized single cells such as human embryonic stem cells) with minor modifications to the protocol.

适用说明：本说明书适用于KAPA HiFi HotStart PCR Kit (KK2500, KK2501及KK2502)。
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适用说明：本说明书适用于KAPA HiFi Uracil+ 热启动高保真酶预混液PCR试验盒 【KK2800(07959044001)， KK2801(07959052001)及 KK2802 (07959079001)】。
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适用说明：本说明书适用于KAPA HiFi PCR Kit (KK2101，KK2102及KK2103)。
友情提示：本网页说明书摘取了原说明书中的产品描述，产品应用及实验操作流程、故障排查等主要信息，欲了解关于更多信息，请参考原说明书 (点击面板上“下载PDF”可下载）。