For each S. aureus isolate, spa types were determined as previously described (75 (link), 76 (link)). Clonal complexes (CCs) were assigned on the basis of spa typing results, using the multilocus sequence typing mapping database (http://www.mlst.net ) or peer-reviewed reports (see Table S2 for a summary of strain isolates). Genomic DNA from S. aureus strain isolates was extracted as previously described (77 (link), 78 (link)), and shotgun sequence data were obtained using 150-bp paired-end reads (Illumina) with ~100× genome coverage.
150 bp paired end reads
Manufactured by Illumina
Sourced in United States
The 150-bp paired-end reads is a core sequencing technology offered by Illumina. It generates high-quality sequencing reads with a length of 150 base pairs per read, and each read is paired with a second read from the opposite end of the DNA fragment.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Novaseq 6000, by Illumina (1 mentions)
Nextseq technology, by Illumina (1 mentions)
Agilent sureselect clinical research exome kit, by Agilent Technologies (1 mentions)
T100 pcr thermal cycler, by Bio-Rad (1 mentions)
Nova 6000 sequencing platform, by Illumina (1 mentions)
Agilent bioanalyzer 2100 dna 1000 kit, by Agilent Technologies (1 mentions)
Nebnext ultra 2 dna prep kit, by New England Biolabs (1 mentions)
7 protocols using 150 bp paired end reads
1
Genomic Profiling of Staphylococcus aureus
2
Single Nuclei Extraction and scDNA-seq
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Individual single nuclei were extracted from frozen tumor tissues of cases S8 and S20 according to the manufacturer’s demonstrated protocol “Isolation of Nuclei for Single Cell DNA Sequencing” (CG000167, Rev A, 10× Genomics). Library preparation of scDNA-seq was performed using Chromium Single Cell DNA Reagent Kits (10× Genomics) according to the instruction (CG000153, Rev C, 10× Genomics). The target cell recovery was set to 500 cells for each sample. Sequencing was conducted using NovaSeq 6000 with 150-bp paired-end reads (Illumina).
3
Estimating Genome Size via k-mer Analysis
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Genome size was estimated using the k-mer analysis of Illumina 150-bp paired-end reads. The k-mer depth-frequency distribution was generated using SOAPec43 (link) (v.2.0.1, https://sourceforge.net/projects/soapdenovo2/ ) with the following parameters: -k 17 -q 33 -t 10. The genome size was then calculated according to the following formula44 (link): genome size = k-mer coverage/mean k-mer depth (Supplementary Table 1 ).
4
Commercial Exome Sequencing for Genetic Variants
5
DNA Extraction and Sequencing of A. japonicus
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The TIANGEN amp Marine Animals DNA Kit (TIANGEN, Beijing, China) was used for the extraction of total genomic DNA in this experiment. A. japonicus muscle was selected instead of the body wall tissue, to avoid the influence of complex organic substances in the body wall, which might affect the quality of nucleic acids. The purity and integrity of the nucleic acids were initially checked by 1% agarose gel electrophoresis, and samples with clear electrophoretic bands and no or mild degradation were qualified. Qualified DNA samples were adjusted to 100 ng/μl and stored at -20 °C.
Qualified DNA samples were randomly fragmented into 350 bp fragments using a Covaris E220 ultrasonic DNA fragmentation machine (Shanghai Tusheng Vision Technology Co., Ltd., Shanghai, China). The library preparation process was completed on a T100 Thermal Cycler PCR (BIO-RAD, USA) instrument using KAPA HyperHlus reagent (Illumina, USA) after 10 cycles of end repair, addition of polyA tails, addition of sequencing adapters, and purification and amplification. High-throughput sequencing of samples was performed using the Illumina Nova6000 sequencing platform (Illumina, California, USA) with a 150 bp paired-end reads (LC-Bio, Hangzhou, Zhejiang Province, China).
Qualified DNA samples were randomly fragmented into 350 bp fragments using a Covaris E220 ultrasonic DNA fragmentation machine (Shanghai Tusheng Vision Technology Co., Ltd., Shanghai, China). The library preparation process was completed on a T100 Thermal Cycler PCR (BIO-RAD, USA) instrument using KAPA HyperHlus reagent (Illumina, USA) after 10 cycles of end repair, addition of polyA tails, addition of sequencing adapters, and purification and amplification. High-throughput sequencing of samples was performed using the Illumina Nova6000 sequencing platform (Illumina, California, USA) with a 150 bp paired-end reads (LC-Bio, Hangzhou, Zhejiang Province, China).
6
Targeted Sequencing of Populus and Salix
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Libraries for two individuals from each Populus balsamifera L., P. tremula L., P. mexicana Wesmael., Salix nigra Marshall, S. exigua Nutt., and S. phlebophylla Andersson (Table S3) were prepared using the NEBNext Ultra II DNA Prep Kit following the published protocol for this kit (New England Biolabs, Ipswitch, MA, USA), and quantified using an Agilent Bioanalyzer 2100 DNA 1000 kit (Agilent Technologies, Santa Clara, CA, USA). Libraries were pooled at equimolar concentrations into two pools of six prior to probe hybridization following the Arbor Biosciences myBaits protocol v 3.0.1 and Hale et al. (2020) . The hybridized samples were subsequently pooled at equimolar ratios and sequenced at the Texas Tech Center for Biotechnology and Genomics using a MiSeq with the Micro chemistry and 150 bp paired-end reads (Illumina, Inc., San Diego, CA, USA).
7
RNA-seq of Drosophila Larval Transcripts
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Total RNA was extracted from late third instar w 1118 , tuSz 1 and hop Tum larvae raised at 28º. In total, three biological replicates were prepared with each biological replicate consisting of 20 pooled larvae. RNA was extracted from whole larvae using Trizol (Thermo Fisher Scientific) followed by QIAGEN RNeasy Micro clean-up (QIAGEN; both according to manufacturer's instructions). RNA concentration was measured using a nanodrop and the integrity was verified using agarose gel electrophoresis. The RNA samples were sent to Novogene (Sacramento, CA) for RNA sequencing (150bp paired end reads, Illumina).
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