Matched genomic DNA (3-5ug) from leukemic and samples at full remission from 56 patients with childhood acute lymphoblastic leukemia (cALL) was prepared for Illumina paired end sequencing (Illumina Inc, SanDiego, CA). Exome enrichment was performed using the Agilent SureSelect Human All Exon 50Mb (Agilent Technologies LTD, Berkshire, UK) kit. Flow-cell preparation, cluster generation and paired end sequencing (75base-pair reads) was performed according to the Illumina protocol guidelines on an Illumina GAII Genome Analyzer. The target coverage per sample was for 70% of the captured regions at a minimum depth of 30× sequencing coverage. Detailed sequencing metrics are provided in Supplementary Table 5 .
Genome analyzer gaii
Manufactured by Illumina
Sourced in Hong Kong, United Kingdom
The Genome Analyzer GAII is a high-throughput sequencing platform developed by Illumina. It is designed to generate large volumes of sequence data for a wide range of genomic applications. The Genome Analyzer GAII utilizes a proprietary sequencing-by-synthesis technology to produce high-quality, accurate DNA sequence data.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000, by Illumina (3 mentions)
Truseq rna sample preparation kit, by Illumina (2 mentions)
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Sureselect human all exon 50 mb, by Agilent Technologies (1 mentions)
Paired end sequencing, by Illumina (1 mentions)
Illustrator cs6, by Adobe (1 mentions)
Seqman ngen, by DNASTAR (1 mentions)
Nebnext rna library preparation kit, by New England Biolabs (1 mentions)
Nextseq 2500, by Illumina (1 mentions)
Clc genomics workbench 6, by Qiagen (1 mentions)
Rna seq sample preparation kit, by Illumina (1 mentions)
Truseq rna sample prep v2 ls protocol, by Illumina (1 mentions)
Qubit rna assay kit, by Thermo Fisher Scientific (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Invitrap spin cell rna mini kit, by Stratec (1 mentions)
11 protocols using genome analyzer gaii
1
Whole Exome Sequencing of Childhood Acute Lymphoblastic Leukemia
2
Genome Sequencing of Bacterial Strains
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The genomes of MB and 11a were sequenced on an Illumina GA-II genome analyzer at the Beijing Genome Institute (BGI-Hong Kong). A paired-end sequencing method of 90 bp long reads in 200 bp insert library was used, yielding coverage depth of about 110 × per genome. The National Center for Bio-technology Information (NCBI) accession numbers for strains MB and 11a are stated in Table 1 . Quality sequences were obtained using a threshold of 20 as quality score47 (link). Detailed methodology on genome construction, open reading frame predictions, annotation, genome comparison, phylogenetic tree construction, heatmap of non-core genes and Clustered regularly interspaced short palindromic repeats (CRISPR) searches can be found as Supplementary Methods online. Adobe Illustrator CS6 was used to enhance the resolution of figures throughout this manuscript.
3
Genome Sequencing of Listeria monocytogenes
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Both L. monocytogenes strains were cultivated under aerobic conditions at 37°C in brain heart infusion broth (BHI, Merck; with 125 rpm shaking) and harvested by centrifugation. The resulting pellet was used for DNA isolation using the QIAGEN genomic-tip columns and buffers (QIAGEN), in accordance with the recommendations of the manufacturer. Genome sequencing was performed using an Illumina GAII genome analyzer at the University of Veterinary Medicine Vienna, Austria. Sequencing was performed using paired-end sequencing technology and 101 bp read-length using Illumina standard protocols. Ten million reads were used for a de novo assembly using SeqManNGen (DNASTAR). For strain QOC1, the assembly of sequenced data resulted in generation of 17 contigs (>500 bp) for which there was an average coverage of 310×, while for strain QOC2, assembly of the reads resulted in 13 contigs (>500 bp), for which the average coverage was 330×. For ordering, the contigs were aligned to the L. monocytogenes EGDe genome using the “move contigs” option in MAUVE [22] (link). Some of the remaining gaps could be closed by PCR and Sanger sequencing - resulting in seven and four contigs in the final assembly for strain QOC1 and QOC2, respectively.
4
Transcriptomic Analysis of N. benthamiana and P. palmivora
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N. benthamiana and P. palmivora messenger RNAs (mRNAs) were purified using Poly(A) selection from the total RNA sample and then fragmented. cDNA library preparation was performed with the TruSeq® RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. cDNA sequencing of the 13 samples (MZ, infected N. benthamiana root samples and uninfected N. benthamiana plants) was performed in four lanes of Illumina NextSeq 2500 in a 100 paired-end mode. Samples were de-multiplexed and analysed further.
mRNAs from additional samples of a short leaf time course (P. palmivora mycelium, N. benthamiana leaves 2 days after inoculation (dai) and N. benthamiana leaves 3 dai) were purified using Poly(A) selection from the total RNA sample. cDNA libraries were prepared using the NEBNext® RNA library preparation kit (New England Biolabs, Hitchin, UK) according to the manufacturer’s protocol and sequenced on an Illumina GAII Genome Analyzer in a 76 paired-end mode in three separate lanes. Reads obtained from these three samples were used for P. palmivora de novo transcriptome assembly only. The raw fastq data are accessible athttp://www.ncbi.nlm.nih.gov/sra/ with accession number SRP096022.
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N. benthamiana and P. palmivora messenger RNAs (mRNAs) were purified using Poly(A) selection from the total RNA sample and then fragmented. cDNA library preparation was performed with the TruSeq® RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. cDNA sequencing of the 13 samples (MZ, infected N. benthamiana root samples and uninfected N. benthamiana plants) was performed in four lanes of Illumina NextSeq 2500 in a 100 paired-end mode. Samples were de-multiplexed and analysed further.
mRNAs from additional samples of a short leaf time course (P. palmivora mycelium, N. benthamiana leaves 2 days after inoculation (dai) and N. benthamiana leaves 3 dai) were purified using Poly(A) selection from the total RNA sample. cDNA libraries were prepared using the NEBNext® RNA library preparation kit (New England Biolabs, Hitchin, UK) according to the manufacturer’s protocol and sequenced on an Illumina GAII Genome Analyzer in a 76 paired-end mode in three separate lanes. Reads obtained from these three samples were used for P. palmivora de novo transcriptome assembly only. The raw fastq data are accessible at
5
Postpartum Milk Transcriptome Analysis
6
Whole-Genome Resequencing of Four Dog Populations
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Four dog populations were collected for this study: 1) Tibetan dogs living in Tibet and Qinghai province at elevations of about 3,500 m; 2) population of indigenous dogs gathered from other provinces of China with elevations lower than 2,000 m; and 3) a population of modern dog breeds (DB) mixed by Chow Chow, Rottweiler, Chihuahua, and Saint Bernard. The genomic DNA was extracted using the Phenol/Chloroform method from peripheral venous blood. For each population, genomic DNA for ten individuals was pooled together equally. Note that 3 chow chows, 3 Rottweilers, 3 Chihuahuas, and 1 Saint Bernard were mixed as one population to represent modern DB. The Illumina Genome Analyzer GAII was used to generate paired-end reads for each population pool according to standard protocols specified by the manufacturer. The read length of raw sequence data was shown on table 1 .
Whole-Genome Resequencing Results for Four Dog Populations
| Populations | Reads Length | Number of Reads (M) | Total Bases | Matching Reads (M) | Matching bp | Effective Depth (Fold) |
|---|---|---|---|---|---|---|
| TID1 | 171 | 317.26 | 54.25 | 292.89 | 38.01 | 15.94 |
| TID2 | 125 | 361.45 | 45.18 | 321.83 | 36.76 | 15.41 |
| Indigenous dog from low-altitude area (CID) | 121 | 248.52 | 30.07 | 209.27 | 21.27 | 8.92 |
| Breed dogs (DB) | 125 | 296.68 | 37.08 | 269.25 | 36.62 | 15.35 |
7
Genomic Profiling of Tuberculosis in Samara
8
Whole Genome Sequencing of Staphylococcus haemolyticus
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WGS was performed on index-tagged libraries (76 bp reads with an average insert size of 400 bp) for each S. haemolyticus strain using an Illumina Genome Analyzer GAII as described in Bentley et al.16 (link) Data have been deposited in the European Nucleotide Archive under the study number ERP000943. Each genome was assembled individually using Velvet17 (link) and contigs >500 bp were ordered relative to the complete reference genome sequence of S. haemolyticus JCSC 1435 (AP006716) using ABACAS.18 (link) Contigs were separated by the spacer sequence 5′-CTAGCTAGCTAG-3′ introducing stop codons in all six reading frames. Protein coding sequences (CDSs) were predicted using Glimmer 3.19 (link) Functional annotations were transferred from the fully sequenced S. haemolyticus JCSC 1435. The unique regions were annotated using an in-house genome annotation pipeline (GePan), combining best functional hits from BLASTp,20 (link) Pfam21 (link) and Priam.22 (link)
9
Genomic Profiling of Tuberculosis in Samara
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From October 2008 to 2010, 2,348 patients with pulmonary disease and culture-proven tuberculosis were recruited from all 18 civilian tuberculosis dispensaries located across Samara. M. tuberculosis isolates were prospectively archived at the Samara Tuberculosis Service, Samara, Russia. Anonymized epidemiological data was stored on a password-protected ACCESS database. Informed consent was obtained from all patients. The study was approved by the Samara Medical Ethics Committee, the Queen Mary Research Ethics Committee and the University of Cambridge Research Ethics Committee.
Isolates were cultured on Middlebrook media for 4–6 wk at 37°C. Sweeps of colonies were harvested, lysed by vortexing with glass beads and genomic DNA purified using a DNeasy Blood and Tissue kit (Qiagen). Paired-end multiplex libraries with a mean insert size of 200bp were prepared as previously described50 (link). Sequencing was performed at the Wellcome Trust Sanger Institute on the Illumina Genome Analyzer GAII or the HiSeq 2000 platform generating reads of 54-bp, 75-bp or 100-bp.
Isolates were cultured on Middlebrook media for 4–6 wk at 37°C. Sweeps of colonies were harvested, lysed by vortexing with glass beads and genomic DNA purified using a DNeasy Blood and Tissue kit (Qiagen). Paired-end multiplex libraries with a mean insert size of 200bp were prepared as previously described50 (link). Sequencing was performed at the Wellcome Trust Sanger Institute on the Illumina Genome Analyzer GAII or the HiSeq 2000 platform generating reads of 54-bp, 75-bp or 100-bp.
10
Mate Pair Sequencing for Structural Variations
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Mate-pair libraries of DNA from case 1, 2 and 3 were prepared using the Mate Pair Library v2 kit (Illumina, San Diego, CA, USA) essentially according to the manufacturer’s protocol. Libraries were quantified and sequenced on an Illumina Genome Analyzer, GAII. Reads were aligned to the human genome using Burrows Wheeler Aligner (BWA)20 (link). Discordant reads were extracted and structural variations were identified using SVDetect21 (link). All genomic coordinates in the study were based on the GRCh37/hg19 assembly.
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