Hiseq 2500 4000

Using the Agilent SureSelect Human All Exome kit (50 Mb), we generated libraries and sequenced them on HiSeq 2500/4000 (Illumina, CA) to generate 2 × 75 bp paired-end data. We obtained a targeted mean coverage of 93x with 93% bases covered at ≥10x (Supplementary Data 2).

ChIP DNA and the input DNA were subjected to end repair and tailing with dA‐tail followed by ligation of adapter sequences. These adapter ligated fragments are then size selected using SPRI bead, followed by size selected fragments are indexed during limited cycle PCR to generate final libraries for paired‐end sequencing. The resulting libraries are quantified and subjected to sequenced on Illumina HiSeq 2500/4000 system to generate 2 × 50 bp sequence reads.

Cytoplasmic RNA samples were submitted to BGI Genomics for selection of polyadenylated RNAs, and strand-specific, paired-end library preparation. During this study, we used two paired-end RNA sequencing methods with the same read depth: DNA nanoball sequencing (DNBseq), which produces 150 bp read lengths, and Illumina sequencing, which produces 100 bp read lengths. We clipped 150 bp DNBseq sample reads to 100 bp and completed the analysis for the 150 bp reads and 100 bp reads in parallel to test whether these read length differences would affect the results. The analysis with different read lengths resulted in a difference of two authentically expressed L1 loci between the groups with 150bp reads producing 14 expressed L1 loci and 100bp reads producing 12 expressed L1 loci. For samples sequenced using Illumina sequencing, samples were pooled in groups of 5–7 and applied to a single lane of an Illumina HiSeq 2500/4000 instrument. Data were sorted based on barcodes attached to each individual sample, aligned to either the Mus musculus 10 (mm10) or Rattus norvegicus 6 (rn6) genomes, and queried for alignments occurring within annotated L1 loci. Bioinformatics analysis was performed as previously described (34–36 (link)). These methods are outlined in more detail below.

The WGS data of 859 participants from the TWB cohort were evaluated using an ultra-fast whole-genome secondary analysis on Illumina sequencing platforms [42 (link)] (Illumina HiSeq 2500/4000). The resulting reads were aligned to the hg19 reference genome with iSAAC 01.13.10.21. iSAAC Variant Caller 2.0.17 was used to perform SNP and insertion-deletion variant discovery and genotyping [42 (link)]. An in-house protocol written in shell script was performed to combine 880 vcf files. A union table of all detected variants in 880 vcf files was used for further analysis. Two gene loci linked to resistin and sST2 levels, according to the GWAS results, were included in the analysis, namely 20 Kb around the RETN and IL1RL1 gene regions. The association between SNPs and resistin and sST2 levels was then analyzed using the GWAS method.

Thirty-one jujube accessions were chosen for genome resequencing analysis, including 10 wild jujube individuals (6 typical wild jujubes and 4 semi-wild accessions) and 21 jujube cultivars (Table O in S2 File; Fig M in S1 File). Illumina paired-end genome libraries were constructed for each accession following the manufacturer’s instructions and then sequenced on Illumina HiSeq 2500/4000 platforms, which yielded a total of 363 Gb of raw paired-end sequences. The raw data were processed to remove low-quality bases, adapter sequences, and putative PCR duplicates, resulting in a total of 344 Gb of high-quality paired-end sequences. The cleaned reads were mapped to the ‘Junzao’ genome using BWA [54 (link)] with the parameters “mem -t 4 -k -M” [29 ]. BAM files were processed for SNP calling using the SAMtools mpileup function [55 (link)] with the parameters “-m 2 -F 0.002 -d 1000 -u.” High-quality SNPs, which were supported by a coverage depth of 5–1,000, mapping quality >20, distance of adjacent SNPs >10 bp and missing ratio of samples within each group <50%, were retained for subsequent analyses.