Next-gen sequencing of assembled gene clusters

Transcription

1 Next-gen sequencing of assembled gene clusters Method Overview Once gene clusters have been assembled in plasmids (either by yeast homologous recombination or using Gibson assembly), plasmid DNA is isolated and the sequences are verified using next-generation sequencing. Plasmid DNA is first fragmented and tagged and then index adapters are attached onto the fragments by PCR. Libraries are then sequenced on Illumina s MiSeq or NextSeq platforms and the resulting short sequence reads are aligned to the expected cluster sequence. Plasmids with SNPs will be removed from further biochemical analysis. We estimate that > 100 plasmids can be sequence-verified in a single sequencing run. Materials: Hard shell pcr plates (E&K Scientific Framestar EK suggested) PCR film (E&K Scientific T796150) Exonuclease V (NEB M0345L) NEB Buffer 4, 10x (NEB P0756S), supplied with Exonuclease V ATP 10 mm (NEB M0440S), supplied with Exonuclease V EDTA, 0.33 M 1.5 mg/ml Sera-mag magnetic particles in PEG/NaCl 10 mm Tris-Cl (ph ) Nextera DNA Sample Preparation Kit, 96 samples (Illumina FC ) Nextera Index Kit, 96 indexes, 384 samples (Illumina FC ) and Additional index primer/adapter set (IDT suggested vendor) Kapa HiFi Hotstart Ready Mix, 2x (Kapa Biosystems KM2602) Sage Science Pippin Prep gel cassette bp Sage Science Pippin Prep 2% marker B Sage Science Pippin Prep loading solution Agilent High Sensitivity DNA kit ( ) KAPA Illumina Platform Library quantification kit (ABI Prism) (KK4835) Equipment: r 96-well magnet plate Sage Science Pippin Prep Instrument capable of small volume quantification such as a Nanodrop ND-1000 or a Qubit fluorometer Agilent Bioanalyzer

2 qpcr instrument Illumina MiSeq Illumina NextSeq High throughput liquid handler (suggested) Protocol Illumina Library Preparation Libraries are typically prepared with samples of plasmid DNA in 96-well plates. Arrange DNA samples in a 96-well plate. Yeast plasmid DNA is treated with Exonuclease V before fragmenting. Exonuclease V: 1.5 µl 10 U/µL Exonuclease V (NEB M0345L) 3.0 µl NEB Buffer 4 (NEB B7004S) 3.0 µl 10 mm ATP (NEB P0756S) 22.5 ul yeast plasmid DNA, 1 µg per 10 U ExoV 37 C, 60 min. 1 µl 0.33 M EDTA 70 C, 30 min. 4.0 C, hold With high throughput liquid hander or manually, clean Exonuclease V treated sample with 1.5 mg/ml Sera-mag magnetic particles. Bead/sample ratio = 1.0. Elute in 25 ul 10 mm Tris-Cl. Prepare PCR master mixes (recipe follows) for index adapter addition in 200 µl strip tubes, a PCR plate, or a 1 ml half block. Master mixes can be made ahead and frozen. Tagmentation: Pre-heat thermal cycler to 55 C. With plate on ice, add together 1.25 µl Nextera TD buffer (Illumina FC kit) 0.25 µl Nextera TDE1 enzyme (Illumina FC kit)

3 1.0 µl ~ 1 20 ng/µl plasmid DNA, equimolar across plate is preferable 55 C, 10 min. Immediately proceed to adapter addition PCR reaction. Adapter addition and library amplification: Make 20 master mixes, 1 for each primer/adapter. for 1 plate: n=12 master mixes 57.2 µl Kapa HiFi Hotstart Ready Mix, 2x (KM2602) 45.6 µl 5 µm Index 1 (700s) n=8 master mixes 85.8 µl Kapa HiFi Hotstart Ready Mix, 2x (KM2602) 68.0 µl 5 µm Index 2 (500s) Add 10 µl of each type of master mix to each well of the tagmentation plate, 20 ul and 2 indexes total added. Final volume = 22.5 µl. 72 C, 3 min. 98 C, 5 min C, 10 sec. 65 C, 30 sec. 72 C, 30 sec. 72 C, 2 min. 4 C, hold Pool together all 22.5 µl of each well. Clean the pooled sample with 1.5 mg/ml Sera-mag magnetic particles. Bead/sample ratio = 1.0. Elute in 60 µl 10 mm Tris-Cl. Size select 30 µl pooled, cleaned DNA between bp, with Sage Science Pippin Prep. Quality check final, size selected library by (1) the Agilent bioanalyzer High Sensitivity DNA assay kit for distribution shape and library size, and (2) qpcr with KAPA Illumina platform library quantification kit or equivalent for concentration.

4 Sequence on Illumina MiSeq system for 192 samples, or the Illumina NextSeq system for 384 samples. Data Processing: The fastq-files are first split into small packages of 100,000 reads in the UNIX console and then processed using an R-script called analyze_fastq.r. This script was run in R version with packages ShortRead and Biostrings installed. In UNIX: # set sample name X=_name_of_miseq_run_ # first split fastq in unix console zcat ${X}_S1_L001_R1_001.fastq.gz split -l xread-1- zcat ${X}_S1_L001_R2_001.fastq.gz split -l xread-2- Copy the miseq_sample_key.txt file to base directory. Then transfer xread files to a new folder /xreads/ in the base directory. Run the R script analyze_fastq.r. Download the analyze_fastq.r script and miseq_sample_key.txt file as a zip file here. References Baym M, Kryazhimskiy S, Lieberman TD, Chung H, Desai MM, Kishony R (2015) Inexpensive Multiplexed Library Preparation for Megabase-Sized Genomes. PLoS ONE 10(5): e doi: / journal.pone MiSeq System User Guide. Illumina part # , catalog # SY DOC Preparing Libraries for Sequencing on the MiSeq. Illumina part # MiSeq Reagent Kit v2 Reagent Preparation Guide. Illumina part # MiSeq Reagent Kit v3 Reagent Preparation Guide. Illumina part # MiSeq Sample Sheet Quick Reference Guide. Illumina part # NextSeq 500 System Guide, Illumina part # Indexed Sequencing Overview Guide, Illumina part # NextSeq System Denature and Dilute Libraries Guide, Illumina part #

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