DNA was extracted from frozen brain tissue of each subject using the DNeasy extraction kit (Quiagen, Valencia, CA). Exome capture was performed on each subject using Nextera enrichment technology (Expanded Exome Oligo kit v4; Illumina, San Diego, CA). This exome capture kit targets the expanded exome, consisting of the 2% of the human genome coding for exons, UTRs and miRNAs. Exome libraries were indexed, and a total of 12 libraries were pooled for high-throughput, 125 bp paired-end sequencing (TruSeq v4 kit) on an Illumina HiSeq 2500 platform. Raw sequencing data were uploaded into BaseSpace (Illumina Inc., CA), a genomic cloud-computing interface. Sequence data of pooled libraries were de-multiplexed using CASAVA v1.8.2 (Illumina), followed by alignment to the human reference genome (build hg19) using the Burroughs-Wheeler aligner (Li and Durbin, 2009 (link)). Next, genotypes were called from aligned sequences following Genome Analysis Toolkit (version 3) best practices (McKenna, et al., 2010 (link)). Quality control steps were performed in PLINK 1.90 (Purcell, et al., 2007 (link)). These included estimation of coverage, call rate, heterozygosity (to rule out contamination), population (to confirm Caucasian ancestry), cryptic relatedness and phenotype-genotype gender matching. None of the samples were excluded based on these stringent quality control metrics.
Casava v1
Manufactured by Illumina
Sourced in United States
CASAVA v1.8.2 is a software application developed by Illumina. It is designed for the analysis of sequencing data generated by Illumina's next-generation sequencing platforms.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000, by Illumina (19 mentions)
Hiseq 2500, by Illumina (16 mentions)
Truseq rna sample preparation kit v2, by Illumina (10 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (7 mentions)
Hiseq 2000 platform, by Illumina (6 mentions)
2100 bioanalyzer, by Agilent Technologies (6 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (6 mentions)
Hiseq 2500 sequencer, by Illumina (5 mentions)
Hiseq 2000 system, by Illumina (5 mentions)
Nebnext ultra rna library preparation kit, by New England Biolabs (5 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Miseq platform, by Illumina (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Mirneasy mini kit, by Qiagen (4 mentions)
Agilent 2100 bioanalyzer dna1000 chip, by Agilent Technologies (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Truseq sbs v3 hs 200 cycle kit, by Illumina (4 mentions)
Rneasy mini and micro kit, by Qiagen (4 mentions)
Rna 6000 nano kit, by Agilent Technologies (3 mentions)
Truseq rna sample prep kit v2, by Illumina (3 mentions)
145 protocols using casava v1
1
Exome Sequencing of Brain Tissue
2
Illumina-Based microRNA Library Preparation
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MiRNA libraries were prepared using Illumina Truseq Small RNA library preparation kit (Illumina, SanDiego, USA) following manufacturer’s protocol. Briefly, 3′ and 5′ adapters were sequentially ligated to the ends of small RNAs fractionated from 1 µg of total RNA, and reverse transcribed to generate cDNA. The cDNA was amplified using a common primer complementary to the 3′ adapter, and a primer containing 1 of 48 index sequences. Samples were size-selected (145–160 bp fragments) on a 6% polyacrylamide gel, purified, quantified and pooled for multiplexed sequencing. The resulting pooled libraries were normalized to 2 nM and were hybridized to oligonucleotide-coated single-read flow cells for cluster generation using HiSeq® Rapid SR Cluster Kit v2 on Hiseq 2500. Subsequently the clustered pooled microRNA libraries were sequenced on the HiSeq 2500 for 50 sequencing cycles using HiSeq® Rapid SBS Kit v2 (50 Cycle). Base calling was performed using CASAVA (v.1.8.2) (Illumina, San Diego, CA, USA) and short-read sequences in FASTQ format were used for downstream analysis.
3
Sequencing Drosophila Ovary Transcriptomes
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All flies for sequencing were reared in 18 C and then transferred to 29 C for either 24 or 96 h to generate pre- and post-tumor conditions, respectively. All flies had access to males and were given yeast supplement for 24 h prior to dissection.
Tissues were dissected from 40 flies in complete medium (Grace’s Insect Basal Medium supplemented with 15% FBS). The anterior section of the ovary was removed. Samples were transferred to a sterile Eppendorf tube and the media was aspirated before freezing samples in liquid nitrogen. Samples were stored until the day of library preparation in −80 C.
Total RNA was extracted using Trizol and libraries were made using the NEBNext Ultra II Directional RNA library Prep Kit for Illumina using the established protocol for use with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB#E7490). We used Rapid Run OBCG single read 50 bp on the Illumina HiSeq 2500 system to sequence these libraries with 2 biological replicates and 2 technical replicates for each time point and genotype. Reads were demultiplexed and indexes removed using CASAVA v1.8.2 (Illumina, USA).
Tissues were dissected from 40 flies in complete medium (Grace’s Insect Basal Medium supplemented with 15% FBS). The anterior section of the ovary was removed. Samples were transferred to a sterile Eppendorf tube and the media was aspirated before freezing samples in liquid nitrogen. Samples were stored until the day of library preparation in −80 C.
Total RNA was extracted using Trizol and libraries were made using the NEBNext Ultra II Directional RNA library Prep Kit for Illumina using the established protocol for use with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB#E7490). We used Rapid Run OBCG single read 50 bp on the Illumina HiSeq 2500 system to sequence these libraries with 2 biological replicates and 2 technical replicates for each time point and genotype. Reads were demultiplexed and indexes removed using CASAVA v1.8.2 (Illumina, USA).
4
Profiling RNA-Protein Interactions in HeLa Cells
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HeLa cells transfected with expression constructs were washed with phosphate-buffered saline (PBS) (−) five times. Total RNA purification and immunoprecipitation (IP) were performed with the RiboCluster Profiler RIP-Assay Kit #RN1001 (MBL International Corporation, Woburn, MA, USA). Immunoprecipitation was carried out using anti-GFP antibody (Clonetech Laboratories, Mountain View, CA, USA) or normal rabbit immunoglobulin G (IgG) as a control. Total RNA was purified with DNase (TURBO™ DNase (0.88 U) (Invitrogen)) and fragmented using mRNA-Seq Sample Prep Kit (Illumina, San Diego, CA, USA). RNA sized 200–400 nt was excised from the acrylamide gel and subject to library preparation. Cluster amplification and single-end sequencing was performed using the Illumina TruSeq SBS Kit v5-GA #FC-104-5001 and Illumina Genome Analyzer GAIIx according to the manufacturer’s protocol (read length: 36 nt). The sequence data was first converted to qseq format by CASAVA v1.8.2 (Illumina), and further converted to FASTQ format using the qseq2fastq converter provided by Kris Popendorf [12 ], and reads containing any base with a Phred quality score of less than 20 were filtered out using FASTX toolkit 0.0.13 [13 ] and custom python code to remove reads containing N’s and homo-polymers consisting solely of one type of nucleotide.
5
RNA Sequencing Library Preparation
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Total RNA was isolated for each sample with Trizol reagent (Life Technologies, Beijing, China) according to the manufacturer’s instruction. The integrity and quality of total RNA samples were analyzed with an RNA 6000 Nano kit (Agilent Technologies, Palo Alto, CA, USA) on a Bioanalyzer 2100 system. Approximately 1 µg total RNA and the Ribo-Zero™ kit (Epicentre, Madison, WI, USA) were used to generate RNA-seq complementary DNA (cDNA) libraries for each sample, and then, RNA sequencing was performed following the manufacturer’s standard procedures. Complementary DNA size and purity were checked using a high sensitivity DNA 1000 kit and AMPure XP beads and was amplified using PCR. High-quality strand-specific libraries were sequenced on the HiSeq X Ten platform (Illumina, San Diego, CA, USA) and the bases were called using the software CASAVA v.1.8.2 (Illumina), then the pair-end reads were acquired.
To avoid artificial bias, we removed low-quality reads including those with ≥10% unidentified nucleotides, >10 nt aligned to the adapter with ≤10% mismatches allowed, and with >50% of bases with phred quality <5. Consequently, 86.46 Gb data was retained, of which the quality of 90% of the base ≥Q30 (Table 1 ). All RNA-seq data have been deposited in NCBI’s (National Center for Biotechnology Information) Gene Expression Omnibus (GEO) series accession number GSE117882.
To avoid artificial bias, we removed low-quality reads including those with ≥10% unidentified nucleotides, >10 nt aligned to the adapter with ≤10% mismatches allowed, and with >50% of bases with phred quality <5. Consequently, 86.46 Gb data was retained, of which the quality of 90% of the base ≥Q30 (
6
Sable Antelope Genome Sequencing
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From the genomic DNA of sable antelope SB#2152, three paired-end libraries with a fragment size of 250 bp and one mate pair library with insert size of ∼5 Kb were prepared using the TruSeq DNA Sample Preparation Kit and the Nextera Mate Pair Library Preparation Kit, respectively, following the manufacturer’s instructions (Illumina, USA). For each library, paired-end sequencing was performed (2 × 101 bp) on a HiSeq 2000. For the five sable antelope provided by the San Diego Zoo Institute for Conservation Research and the two individuals from the wild, a paired-end library (200-500 bp) was constructed for each individual using the TruSeq DNA Sample Preparation Kit (Illumina, USA) and sequenced on a HiSeq2000 or HiSeq1500. Sequencing reads were processed using CASAVA v1.8.2 (Illumina, USA).
7
Illumina HiSeq 2000 Paired-End Sequencing
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The libraries were sequenced on an Illumina HiSeq 2000 platform using a 101-bp paired-end sequencing strategy. The original image data generated by the sequencing machine were converted into sequence data via base calling (Illumina pipeline CASAVA v1.8.2) and then subjected to standard quality control (QC) criteria to remove all of the reads that fit any of the following parameters: (1) reads that aligned to adaptors or primers with no more than two mismatches, (2) reads with >10% unknown bases (N bases), and (3) reads with >50% of low-quality bases (quality value ≤5) in one read. Finally, 1308.3 Gb (94.4%) of filtered reads were left for further analysis after QC.
8
Conjunctival Impression Cytology DNA Sequencing
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A total of 100 ng DNA from conjunctival impression cytology samples was sonicated into fragments of 300 to 400 bp using Bioruptor (Diagenode, Seraing, Belgium) and subjected to sequencing library preparation following the standard protocol provided by the manufacture using KAPA LTP Library Preparation Kit (Kapa Biosystems, Wilmington, MA, USA). DNA libraries were sequenced to a depth of ∼ 50 million reads per sample using HiSeq PE Cluster Kit v4 and HiSeq SBS V4 250 cycle kit (Illumina, San Diego, CA) on the Illumina HiSeq2500 sequencer and subjected to initial processing using CASAVA (v1.8.2) (Illumina). All five blank samples were subjected to the same sequencing library preparation process.
9
CY12 Genome Sequencing Workflow
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Raw sequencing data were generated by Illumina base-calling software CASAVA v1.8.2 (http://support.illumina.com/sequencing/sequencing_software/casava.ilmn (accessed on 18 September 2021)) according to its corresponding manuscript. Trimmomatic (http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic (accessed on 18 September 2021)) uses default parameters to identify contamination reads, such as those containing adaptors or primers. Clean data obtained by the above quality control processes were further analyzed. The CY12 genome was sequenced using a combination of the PacBio RS and Illumina sequencing platforms. The Illumina data were used to assess genomic complexity and correct the PacBio long reads.
10
GBS Library Preparation and Sequencing
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Library preparation and high-throughput sequencing were performed at University of Wisconsin Biotechnology Center (DNA Sequencing Facility). The GBS genomic library preparation was done following the protocol detailed by Elshire et al. [18 (link)] with the methylation-sensitive restriction enzyme ApeKI and 96 custom barcodes. Illumina high-throughput sequencing was conducted on an Illumina HiSeq 2000 (Illumina, USA) using 100 bp single-end sequencing runs. The samples were sequenced across one Illumina lane. Base calling was performed in Casava v1.8.2 (Illumina, USA).
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