Frozen mycelia of all isolates were ground in liquid nitrogen and genomic DNA was extracted as previously described [65 ,66 ]. Standard techniques for manipulation of DNA were used [67 ]. The genomes of all clinical isolates and the type strain of A. latus (9 in total) were sequenced at the Genomic Services Lab of Hudson Alpha (Huntsville, Alabama, USA) on an Illumina HiSeq 2500 sequencer; the sole exception was A. quadrilineatus NRRL 201T, which was sequenced using on a NovaSeq S4 at the Vanderbilt Technologies for Advanced Genomes facility (Nashville, Tennessee, USA). All isolates were sequenced using paired-end sequencing (150 bp) with the Illumina TruSeq library kit. Additionally, the type strain of A. latus and the clinical isolates MM151978 and NIH were also sequenced using mate-pair sequencing (150 bp) using the Illumina Nextera Mate Pair Library kit with an insert size of 4 kilobases. The genome coverage of each isolate was greater than 150X. Both the raw short-read sequence data and the genome assemblies are publicly available (see Table S3 for accession numbers).
Nextera mate pair library kit
Manufactured by Illumina
The Nextera mate-pair library kit is a laboratory equipment product designed to prepare mate-pair libraries for DNA sequencing. The kit enables the construction of libraries with long-range genomic information, which can be useful for genome assembly and structural variation detection.
Automatically generated - may contain errors
3 protocols using nextera mate pair library kit
1
Whole-Genome Sequencing of Aspergillus Isolates
2
High-Molecular-Weight DNA Extraction and Sequencing
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We extracted high molecular weight (HMW) DNA from fresh tissue ground in a bead beater with PVP buffer. We used a modified Qiagen Puregene Gentra kit protocol (S1 ) for HMW DNA extraction. We assessed DNA concentration with a Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA) and ran an agarose gel to assess DNA fragment integrity and size. An Illumina DNA TruSeq library was created for regular whole-genome shotgun sequencing. Mate-pair libraries were created with 6 kbp insert sizes using an Illumina Nextera mate-pair library kit. Additional flash-frozen leaf tissue was sent to Dovetail Genomics (Scotts Valley, CA) for Hi-C and Chicago library preparation and sequencing.
3
Genome Assembly of Aspergillus spinulosporus
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Mycelia were grown on potato dextrose agar for 72 h before lyophilization. Lyophilized mycelia were lysed by grinding in liquid nitrogen and suspension in extraction buffer (100 mM Tris-HCl, pH 8, 250 mM NaCl, 50 mM EDTA, and 1% SDS). Genomic DNA was isolated from the lysate with a phenol-chloroform extraction followed by an ethanol precipitation.
DNA was sequenced with both paired-end and mate-pair strategies to generate a high-quality genome assembly. Paired-end libraries and mate-pair libraries were constructed at the Genomics Services Lab at HudsonAlpha (Huntsville, AL) and sequenced on an Illumina HiSeq X sequencer. Paired-end libraries were constructed with the Illumina TruSeq DNA kit, and mate-pair libraries were constructed with the Illumina Nextera mate-pair library kit targeting an insert size of 4 kb. In total, 63 million paired-end reads and 105 million mate-pair reads, each of which was 150 bp in length, were generated.
TheAspergillus spinulosporus genome was assembled using the iWGS pipeline (102 (link)). Paired-end and mate-pair reads were assembled with SPA des, version 3.6.2 (103 (link)), using optimal k-mer lengths chosen using KmerGenie, version 1.6982 (104 (link)), and evaluated with QUAST, version 3.2 (105 (link)). The resulting assembly is 33.8 Mb in size with an N50 of 939 kb.
DNA was sequenced with both paired-end and mate-pair strategies to generate a high-quality genome assembly. Paired-end libraries and mate-pair libraries were constructed at the Genomics Services Lab at HudsonAlpha (Huntsville, AL) and sequenced on an Illumina HiSeq X sequencer. Paired-end libraries were constructed with the Illumina TruSeq DNA kit, and mate-pair libraries were constructed with the Illumina Nextera mate-pair library kit targeting an insert size of 4 kb. In total, 63 million paired-end reads and 105 million mate-pair reads, each of which was 150 bp in length, were generated.
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