The obtained genomic DNA was then submitted to the Illumina high-throughput sequencing (samples S1 and S7) and 454 pyrosequencing (samples S1–S8), which were performed using the Illumina Hiseq 2000 and FLX Titanium platform of Roche 454 from the Beijing Genome Institute (Shenzhen, China), respectively. The bacterial 16S rRNA gene (V3-V4 region, approximately 460 bp) was carried out with Illumina-specific fusion primers V3F and V4R (Claesson et al., 2009 (link)). PCR amplification was conducted with previous report (Huang et al., 2014 (link)). The 12-bp barcode with primers was used to assign individual sequences to samples. The 16S rRNA gene amplicons were submitted to an Agilent 2100 Bioanalyzer (Agilnet, United States) before using FLX Titanium platform of 454 pyrosequencing (Roche, United States). The GenBank accession numbers for the genomic datasets in NCBI are SRX825942 and SRX825518 .
Hiseq 2000
Manufactured by Roche
Sourced in China, United Kingdom
The HiSeq 2000 is a high-throughput DNA sequencing system designed for large-scale genomic research. It utilizes sequencing-by-synthesis technology to generate DNA sequence data. The HiSeq 2000 is capable of producing high-quality, paired-end reads with flexible read lengths and throughput options.
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Lab products found in correlation
Hiseq 2000, by Illumina (2 mentions)
High throughput sequencing, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Rnase a, by Qiagen (1 mentions)
Gs flx, by Roche (1 mentions)
Genome analyzer iix, by Illumina (1 mentions)
Mrna sequencing kit, by Illumina (1 mentions)
454 genome sequencer flx, by Roche (1 mentions)
Sequencing platform, by Illumina (1 mentions)
8 protocols using hiseq 2000
1
Illumina and 454 Sequencing of Bacterial 16S rRNA
2
Mantle Tissue Transcriptomic Assembly
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Seven total RNA extractions derived from the distal-most edge (i.e. the shell forming region) of the mantle tissue of 4 juvenile C. nemoralis individuals (two extractions from each of three individuals for Illumina sequencing and one extraction from a fourth individual for 454 sequencing) was extracted using Trizol according to the manufacturer’s instructions. These total RNA extractions were sequenced on the Illumina HiSeq2000 and Roche 454 platforms and assembled using the CLC Genomics workbench (version 5.5.2). This assembly generated a total of 676,358 contigs >100 bp, summing to a total of 193,892,905 bp (max contig size = 14,765 bp, median contig size = 180 bp). In order to reduce the redundancy of this assembly for proteomic interrogation (see below) the following steps were applied. First, contigs shorter than 500 bp were removed. All remaining contigs were then clustered into isotigs using “usearch” [59 (link)]. The longest possible open reading frame (which was required to be >50 amino acids) was then extracted from each isotig using standard Perl scripts. These translated ORFs were then clustered again using “usearch” to produce a total of 55,623 putative coding translated fragments. This dataset was used to conduct the proteomic surveys (see below). All assembled nucleic acid sequences discussed in this work are available in Additional file 11 .
3
High-quality Genome Assembly of O. sinensis
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We sequenced the O. sinensis genome by using a combination of Illumina HiSeq 2000 and Roche 454 sequencing technologies. The obtained reads were assembled using Newbler16 (link) and SSPACE17 (link), yielding a high-quality genome assembly. We assessed the quality of the assembled genome by using four evaluation methods. Firstly, we aligned all DNA and EST sequences of O. sinensis available in public databases to the final genome assembly and calculated mapping rates. Secondly, we mapped all clean sequencing reads to the assembled genome sequences and statistically examined mapping rates of these reads to the whole genome. Third, we assembled RNA sequencing data of O. sinensis generated in this study into transcripts using Trinity, and then further evaluated the assembly quality by aligning these assembled transcripts against the sequences from the final assembly of O. sinensis genome. Finally, we mapped the BUSCO conserved genes from Ascomycota lineage to our genome assembly and then analyzed the mapping result.
4
Whole-genome Shotgun Sequencing of CHM1
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We performed whole-genome shotgun sequencing on the CHM1 DNA. KAPA qPCR (KK4854, Kapa Biosystems) was performed on the LightCycler 480 System (Roche Life Sciences) to quantify the libraries and determine the appropriate concentration to produce optimal recommended cluster density on a HiSeq 2000 V2 or V3 2 × 100-bp sequencing run. HiSeq 2000 V2 and V3 runs were completed according to the manufacturer’s recommendations (Illumina). We generated > 617 Gb of sequence used for the assembly. The average insert size was 315 bp for three libraries, 3 kbp for three libraries, and 8 kbp for two libraries.
5
Comparative Analysis of M2 Chromosome in S. aureus
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Shotgun sequencing of the M2 chromosomal DNA was performed using Illumina HiSeq 2000 (single reads) and Roche 454 (8-kb paired-end library). All reads were incorporated into a hybrid assembly using Newbler v2.6. Scaffolds were scanned for open reading frames (ORFs) and annotated using RAST [15 ] and BLAST [16 ].
The resulting M2 chromosome scaffold was compared to the published ST5 S. aureus reference complete genomes (N315, Mu3, Mu50) using snpTree-1.1 [17 ] to construct a SNP phylogenetic tree. We compared the M2 and N315 genomes for homologs using BlastP bidirectional best hit analysis with an identity threshold of 60% and an E-value threshold of 1E-08.
The resulting M2 chromosome scaffold was compared to the published ST5 S. aureus reference complete genomes (N315, Mu3, Mu50) using snpTree-1.1 [17 ] to construct a SNP phylogenetic tree. We compared the M2 and N315 genomes for homologs using BlastP bidirectional best hit analysis with an identity threshold of 60% and an E-value threshold of 1E-08.
6
Sequencing of Naegleria fowleri Genome
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DNA was extracted from 108
N. fowleri trophozoites cultivated in Nelson’s medium using the DNeasy Blood and Tissue Kit (Qiagen, Basel, Switzerland) according to the manufacturer’s protocol. To obtain RNA-free DNA, RNA digestion was performed using 4 μl of RNase A (Qiagen). The DNA was eluted with 100 μl of 10 mM TrisHCl, pH 8.5, pre-heated to 70°C. The DNA quality was visualized on 0.8% agarose gels, and quantification was performed using a NanoDrop® device. Three micrograms of high-molecular weight DNA was sent to Fasteris (Plan-les-Quates, Switzerland) for paired-end sequencing, with an insert size of 300 bp, using the Illumina HiSeq 2000 platform, while 20 μg of DNA was sent to GATC Biotech (Constance, Germany) for preparation of a 3-kb mate-pair library using Illumina technologies and for Roche 454 GS FLX sequencing.
The NGS reads have been deposited in DDBJ/EMBL/GenBank under accession SRX523949 (Illumina HiSeq 2000 reads) and SRX547942 (Roche 454 GS FLX reads).
N. fowleri trophozoites cultivated in Nelson’s medium using the DNeasy Blood and Tissue Kit (Qiagen, Basel, Switzerland) according to the manufacturer’s protocol. To obtain RNA-free DNA, RNA digestion was performed using 4 μl of RNase A (Qiagen). The DNA was eluted with 100 μl of 10 mM TrisHCl, pH 8.5, pre-heated to 70°C. The DNA quality was visualized on 0.8% agarose gels, and quantification was performed using a NanoDrop® device. Three micrograms of high-molecular weight DNA was sent to Fasteris (Plan-les-Quates, Switzerland) for paired-end sequencing, with an insert size of 300 bp, using the Illumina HiSeq 2000 platform, while 20 μg of DNA was sent to GATC Biotech (Constance, Germany) for preparation of a 3-kb mate-pair library using Illumina technologies and for Roche 454 GS FLX sequencing.
The NGS reads have been deposited in DDBJ/EMBL/GenBank under accession SRX523949 (Illumina HiSeq 2000 reads) and SRX547942 (Roche 454 GS FLX reads).
7
Analyzing Somatic Mutations in FAT1 and TP53
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For the TCGA cohort, whole‐exome somatic mutation data released on October 12, 2012, were downloaded from the TCGA data portal.13 Whole‐exome sequencing was performed using the Illumina Genome Analyzer IIX platform. For the ICGC cohort, whole‐exome somatic mutation data released on September 12, 2014, were obtained from the ICGC data portal.14 Whole‐exome sequencing was performed using the Illumina HiSeq 2000 and Roche GS‐FLX platforms, and some of the mutations that were sequenced only on one platform were verified using Ion Torrent PGM. Detailed information on the sequencing, quality control, raw data processing, and validation procedure is provided elsewhere.13 , 14 After downloading the data, the somatic mutation profile of FAT1 and TP53 was analyzed for each tumor. Nonsense mutations, frameshift indels, and splice‐site mutations were considered as loss‐of‐function mutations. For subsequent analysis, patients were categorized into 2 groups according to their FAT1 mutational status: those with mutant FAT1 (ie, presence of at least one nonsynonymous FAT1 mutation), and those with wild‐type FAT1 (ie, no nonsynonymous FAT1 mutations).
8
Shotgun Sequencing and RNA-Seq Library Construction
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Genomic DNA for shotgun sequencing was sheared with average fragment sizes of 500 bp, 3 kb, and 10 kb. Three DNA libraries were constructed using the Illumina sequencing platform according to the manufacturer’s protocol (Illumina CA, US). Briefly, 5, 10, and 20 μg of genomic DNA were used for whole genome random shearing to construct 500 bp, 3 kb, and 10 kb libraries, respectively. Fragments were ligated with 3′ and 5′ adapters to produce libraries for PCR amplification.
For mRNA sequencing, cDNA libraries were prepared for each tissue using the Illumina mRNA sequencing kit (Illumina, CA, US) and the Clontech SMART cDNA Library Construction Kit (Invitrogen). Libraries were sequenced using the Illumina HiSeq2000 and Roche 454 Genome Sequencer FLX instrument (Roche, UK).
For mRNA sequencing, cDNA libraries were prepared for each tissue using the Illumina mRNA sequencing kit (Illumina, CA, US) and the Clontech SMART cDNA Library Construction Kit (Invitrogen). Libraries were sequenced using the Illumina HiSeq2000 and Roche 454 Genome Sequencer FLX instrument (Roche, UK).
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