全基因组测序和分析Whole genome sequencing (WGS) and analysis

使用改良的SDS/蛋白酶K方法提取噬菌体裂解液的基因组DNA。简单地说,将200μL高滴度噬菌体裂解液(>109 pfu/ml)与等体积的裂解缓冲液(10 mM Tris, 10 mM EDTA, 100 μg/mL蛋白酶K, 100 μg/mL RNaseA, 0.5% SDS)混合,在37℃下孵育30分钟,然后在55℃下孵育30分钟。使用DNA清洁和浓缩试剂盒(Zymo Research)进一步纯化预处理。使用Qubit 4.0荧光仪(Life Technologies)对DNA进行定量。使用Illumina DNA Prep Kit(以前称为Illumina Nextera Flex Kit)制备WGS文库,使用修改后的方案,每次预处理使用的标记试剂数量减少了5倍,除了用Tagment Wash Buffer(TWB)洗珠步骤,其中使用了推荐的100μL TWB。随后的珠上PCR索引扩增是使用2倍Phusion Master Mix(NEB)和定制的索引引物(IDT)进行的,与Illumina Nextera Index Kit的序列匹配。每个50μL的反应被分成两管,分别扩增9和12个循环。库通过琼脂糖凝胶电泳进一步纯化;DNA在400bp大小的范围内被切除,并使用Zymoclean Gel DNA Recovery Kit(Zymo Research)进行纯化。库用Qubit进行定量,使用9周期反应,除非产率太低无法测序,在这种情况下使用12周期反应。库以等摩尔比例汇集,用Illumina MiSeq v3试剂测序(150个循环,读数1;8个循环,指数1;8个循环,指数2)。WGS数据在仪器上或使用定制的解复用Python脚本(由Nimit Jain博士编写)进行解复用,并使用cutadapt(v 3.462)进行修剪以去除Nextera适配器。使用Bowtie 2.0 (-very-sensitive-local alignments63)对修剪后的读数进行映射,并使用IGV (v 2.9.472)对排列进行了可视化。使用SeqDiff程序(https://github.com/hansenlo/SeqDiff)检测变异。

Genomic DNA from phage lysates was extracted using a modified SDS/Proteinase K method. Briefly, 200 μL high titer phage lysate (>109 pfu/ml) was mixed with an equal volume of lysis buffer (10 mM Tris, 10 mM EDTA, 100 μg/mL proteinase K, 100 μg/mL RNaseA, 0.5% SDS) and incubated at 37°C for 30 min, and then 55°C for 30 min. Preps were further purified using the DNA Clean & Concentrator Kit (Zymo Research). DNA was quantified using the Qubit 4.0 Fluorometer (Life Technologies). 20-100 ng genomic DNA was used to prepare WGS libraries using the Illumina DNA Prep Kit (formerly known as Illumina Nextera Flex Kit) using a modified protocol that utilized 5x reduced quantities of tagmentation reagents per prep, except for the bead washing step with Tagment Wash Buffer (TWB), where the recommended 100 μL of TWB was used. Subsequent on-bead PCR indexing-amplification of tagmented DNA was performed using 2x Phusion Master Mix (NEB) and custom-ordered indexing primers (IDT) matching the sequences from the Illumina Nextera Index Kit. Each 50 μL reaction was split in two tubes, amplified for 9 and 12 cycles respectively. Libraries were further purified by agarose gel electrophoresis; DNA was excised around the ∼400 bp size range and purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research). Libraries were quantified by Qubit and the 9-cycle reaction was used unless the yield was too low for sequencing, in which case the 12-cycle reaction was used. Libraries were pooled in equimolar ratios and sequenced with Illumina MiSeq v3 reagents (150 cycles, Read 1; 8 cycles, Index 1; 8 cycles, Index 2). WGS data were demultiplexed either on-instrument or using a custom demultiplexing Python script (written by Dr. Nimit Jain), and trimmed using cutadapt (v 3.462) to remove Nextera adapters. Trimmed reads were mapped using Bowtie 2.0 (–very-sensitive-local alignments63) and alignments were visualized using IGV (v 2.9.472). Variants were detected using the SeqDiff program (https://github.com/hansenlo/SeqDiff).

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