One S. pilchardus specimen (Figure 1 ) was caught off Esposende (41.501944 N 8.851667 W), Portugal, under the “Programa Nacional de Amostragem Biológica” carried out by the Instituto Português do Mar e da Atmosfera (IPMA). Tissues were harvested immediately and stored in 100% ethanol (muscle) and RNA later (liver) until further processing (Supplementary Table S1 in Supplementary File 2 ). Genomic DNA was extracted from muscle tissue (~0.5 g) in three replicates, using Qiagen’s DNeasy Blood & Tissue Kit (Valencia, CA, USA) according to the manufacturer’s instructions, with the following modifications: prior to elution in 100 μL AE buffer, samples were incubated at 37 °C for 10 min, to increase DNA yield. DNA concentration and integrity were verified using an Agilent Genomic DNA ScreenTape (Waldbronn, Germany). We constructed one 150 bp paired-end reads library from 1.2 μg of genomic DNA using the standard Illumina protocol for the TruSeq Nano DNA library kit (Illumina, San Diego, CA, USA). Sequencing was performed with the Illumina HiSeq X Ten (Macrogen, Seoul, Korea) platform and generated 69.0 Gbp of raw reads for downstream analysis (Supplementary Table S1 in Supplementary File 2 ).
Hiseq x ten
Manufactured by Macrogen
Sourced in Japan
The HiSeq X Ten is a high-throughput DNA sequencing system manufactured by Illumina. It is designed to perform large-scale human genome sequencing at a high speed and efficiency. The HiSeq X Ten is capable of sequencing multiple human genomes in a single run, making it a valuable tool for genomic research and clinical applications.
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Lab products found in correlation
Miseq reagent kit v3, by Illumina (3 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
Nebnext library quant kit, by New England Biolabs (2 mentions)
Truseq nano dna library kit, by Illumina (1 mentions)
Genomic dna screentape, by Agilent Technologies (1 mentions)
Miseq system, by Illumina (1 mentions)
Reagent kit version 3, by Illumina (1 mentions)
Novaseq 6000, by Macrogen (1 mentions)
Unmethylated lambda dna, by Promega (1 mentions)
Truseq nano dna library preparation kit, by Illumina (1 mentions)
Library quantification kit, by Takara Bio (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
Library quantitation kit, by Takara Bio (1 mentions)
Nebnext ultra 2 directional rna library prep kit, by New England Biolabs (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Rna 6000 nano kit, by Agilent Technologies (1 mentions)
Qiazol, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nanodrop 1, by Thermo Fisher Scientific (1 mentions)
14 protocols using hiseq x ten
1
Genomic DNA Extraction and Sequencing of Sardine (Sardina pilchardus)
2
Illumina Paired-end Sequencing Library Preparation
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To prepare sequencing library for SR sequencing, DNA was randomly sheared by sonication and 300–500 bp fraction was isolated by gel excision and used for Illumina paired-end (PE) following the instructions provided by the manufacturers. SR libraries used in this work include (1) 2 × 90 bp PE SR library, sequenced by HiSeq 2000 (BGI); (2) 2 × 150 bp PE SR library, sequenced by HiSeq X Ten (Macrogen Inc.); and (3) 2 × 150 bp mate-pair (MP) library with insert size of ~ 3 kb was also prepared and sequenced (BGI).
SR raw reads were processed by the following procedure. Raw reads were examined with FastQC33 (link) for base quality and the presence of adapters. AdapterRemoval tool34 (link),35 (link) was then used to remove the adapter sequences if present. Terminal ambiguous bases were trimmed off by Ambiguity trimming module of NGS QC Toolkit, and low quality reads were filtered out using IlluQC module of NGS QC Toolkit36 (link). Reads > = 100 bp were selected by PRINSEQ software37 (link). Mapping to reference genome was performed by BWA38 (link). The insert size of MP reads was estimated by BamTools-based script and reads with insert sizes of about 2600 (bp) +/− 30% were selected and used in assembly.
SR raw reads were processed by the following procedure. Raw reads were examined with FastQC33 (link) for base quality and the presence of adapters. AdapterRemoval tool34 (link),35 (link) was then used to remove the adapter sequences if present. Terminal ambiguous bases were trimmed off by Ambiguity trimming module of NGS QC Toolkit, and low quality reads were filtered out using IlluQC module of NGS QC Toolkit36 (link). Reads > = 100 bp were selected by PRINSEQ software37 (link). Mapping to reference genome was performed by BWA38 (link). The insert size of MP reads was estimated by BamTools-based script and reads with insert sizes of about 2600 (bp) +/− 30% were selected and used in assembly.
3
Microbiome Identification Workflow
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Sequences were determined by 150 × 150 PE sequencing (30×) using Illumina HiSeq X Ten (Macrogen Corp).
Illumina sequencing adapter was trimmed by cutadapt. Both base quality and read quality were checked after contaminant reads were removed. PRINseq [27 (link)] was employed for base quality checking following the sequential steps: removal of low quality bases from both 5′- and 3′-ends, removal of reads having ≥ 3 ambiguous (N) bases, and removal of reads with read length < 30 bp. NGS QC Toolkit [28 (link)] was then used to select high quality reads each of which has base score ≥ 20 in ≥ 70% of the contained bases. A generalized workflow for data analysis is shown in Fig.1 .![]()
Illumina sequencing adapter was trimmed by cutadapt. Both base quality and read quality were checked after contaminant reads were removed. PRINseq [27 (link)] was employed for base quality checking following the sequential steps: removal of low quality bases from both 5′- and 3′-ends, removal of reads having ≥ 3 ambiguous (N) bases, and removal of reads with read length < 30 bp. NGS QC Toolkit [28 (link)] was then used to select high quality reads each of which has base score ≥ 20 in ≥ 70% of the contained bases. A generalized workflow for data analysis is shown in Fig.
Workflow showing the stepwise procedure of sequence data processing leading to the identification of microbes in the body
4
Arabidopsis Methylome Profiling by Bisulfite Sequencing
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All Sequencing procedures were performed using the HiSeqXten platform (Macrogen, Korea). Paired end reads (150 bp) were generated. All reads were trimmed (10 bp for 5’end and 5 bp for 3’end) using Trim galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/ ), and low quality and short reads (<70 bp) were removed using Trimmomatic (http://www.usadellab.org/cms/?page=trimmomatic ). Reads were mapped to the Arabidopsis TAIR10 genome by hisat2 (http://daehwankimlab.github.io/hisat2/ ) using Bismark (https://www.bioinformatics.babraham.ac.uk/projects/bismark/ ) under the option –hisat2 –local. PCR duplicates were removed and methylation levels were extracted using the Bismark toolset (deduplicate_bismark and bismark_methylation_extractor, respectively). Read counts for all cytosine methylation contexts (CpG, CHG, and CHH) were calculated by how many reads were mapped on each context (1 bp resolution). Methylation levels were basically calculated by dividing the counts of methylated cytosine by the number of cytosine (meC + C). Except for genomic features, we used 50 bp windowed average having more than each three cytosine context with at least five reads.
5
Small and Large-scale Sequencing Protocols
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Small-scale sequencing was performed using the Illumina MiSeq system with the reagent kit version 3 (150 cycles) and the reagent nano kit version 2 (300 cycles) (San Diego, CA, USA), with some modifications for denaturation and neutralization of the library, as described previously (8 (link)). The amount of input library varied depending on the purpose of the experiments. Large-scale sequencing using the HiSeq X Ten and NovaSeq 6000 were performed by Macrogen Japan Corp. (Kyoto, Japan) as commercial services. Sequenced reads were delivered to us after demultiplexing of indexed libraries.
6
Whole-Genome Bisulfite Sequencing of Hepatocytes and Skeletal Muscle
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Genomic DNAs from hepatocytes and skeletal muscle was prepared using DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The library preparation for WGBS was performed with the tPBAT protocol described previously72 (link). In brief, 100 ng of purified genomic DNA was spiked with 1 ng of unmethylated lambda DNA (Promega), and the mixture was bisulfite converted. Then, the bisulfite-treated DNA was split into two portions and individually served for library preparations in forward and reverse directions. The two libraries of different directions were mixed and served for sequencing. This mixed library strategy is effective for signal complementation of biased nucleotide composition of WGBS reads72 (link). Sequencing was performed with HiSeq X ten at Macrogen Japan (Tokyo, Japan), assigning a lane per sample.
We used transcriptome of WT mice under the same condition as this study obtained in our previous studies22 (link),23 , but instead of FPKM (fragments per kilobase of exon per million reads mapped), TPM (transcript per million) was used as the gene expression level.
We used proteome of WT mice under the same condition as this study measured with iBAQ-MS obtained in our previous study23 .
We used transcriptome of WT mice under the same condition as this study obtained in our previous studies22 (link),23 , but instead of FPKM (fragments per kilobase of exon per million reads mapped), TPM (transcript per million) was used as the gene expression level.
We used proteome of WT mice under the same condition as this study measured with iBAQ-MS obtained in our previous study23 .
7
Robust Genomic DNA Sequencing Protocol
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Genomic DNA extracted from blood was assessed for quality by PicoGreen and gel electrophoresis. Library preparation was done in-house (The University of Hong Kong, Centre for Genomic Sciences) using TruSeq Nano DNA library preparation kit and then sequenced at 30X coverage using Illumina HiSeqX Ten (150-bp paired-end sequencing) by Macrogen. The paired-end sequence reads were processed according to the GATK best practice recommendations (DePristo, Banks et al. 2011) . Sequencing output metrics are detailed in Table S2. Variants, including SNPs and insertions/deletions (indels), were called by GATK Haplotype Caller (v3.4) and variant quality recalibration was performed as variant-based quality control.
We further performed genotype-based quality control by restricting the analysis to high quality calls with genotype quality (GQ)>20 and depth (DP)>8 using KGGseq (Li, Li et al. 2017) .
We further performed genotype-based quality control by restricting the analysis to high quality calls with genotype quality (GQ)>20 and depth (DP)>8 using KGGseq (Li, Li et al. 2017) .
8
High-throughput Illumina Sequencing
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Small-scale sequencing was performed using Illumina MiSeq with MiSeq Reagent Kit v3 (150 cycles) in the paired-end mode of 2× 75 cycles. For large-scale sequencing, paired-end sequencing with 2× 150 cycles using the HiSeq X Ten was performed by Macrogen Japan Corp. (Kyoto, Japan). The reads were delivered after demultiplexing, and indexed libraries were used for subsequent bioinformatics analysis.
9
Paired-end Illumina Sequencing Protocol
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Small-scale sequencing was performed using Illumina MiSeq with MiSeq Reagent Kit v3 (150 cycles) in the paired-end mode of 2× 75 cycles. For large-scale sequencing, paired-end sequencing with 2× 150 cycles using the HiSeq X Ten was performed by Macrogen Japan Corp. (Kyoto, Japan). The reads were delivered after demultiplexing, and indexed libraries were used for subsequent bioinformatics analysis.
10
Sequencing Library Quantification and Scaling
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The molar concentration of the sequencing library was determined using the library quantification kit from Takara Bio Inc. Small-scale sequencing was performed with MiSeq using the MiSeq Reagent Kit v2 nano kit (300 cycles) in the paired-end mode with 2 × 151 cycles. For large-scale sequencing, the paired-end mode with 2 × 151 cycles using the HiSeq X Ten was performed by Macrogen Japan Corp. (Tokyo, Japan).
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