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SRX9187349: TB-13
1 ILLUMINA (Illumina HiSeq 2500) run: 793,196 spots, 198.3M bases, 76.6Mb downloads

Design: Genomic DNA was extracted using Qiamp extraction kit. Truseq Nano DNA library preparation method was used for sequencing. Covaris E220 was used to shear the DNA. The sequenced has been done using the Hiseq 2500 with high-output mode (2x125bp) and the Miseq with v3 (2x300bp) reagents. TruSeq Nano DNA Library Prep Workflow Quantify gDNA was done using a fluorometric-based method. Normalize gDNA samples with RSB to a final volume of 52.5 l in the DNA plate, 100 ng for a 350 bp insert size was used, 200 ng for a 550 bp insert size 3 Mix thoroughly was done. Then Shake at 1800 rpm for 2 minutes. Pipette up and down and 4 Centrifuged was done as follows, Centrifuge at 280 g for 1 minute. [LS] Centrifuge briefly. Then 52.5 l DNA samples was transferred to separate Covaris tubes. The wells of the CFP plate were used to hold Covaris tubes upright. 2 Centrifuge at 280 g for 5 seconds. 3 Fragment the DNA using the following Covaris settings. Centrifuge at 280 g for 5 seconds was done and then transfer 50 l supernatant from each Covaris tube to the corresponding well of the CSP plate. Clean Up Fragmented DNA The SPB was vortexed until well-dispersed and 80 l SPB to each well, and then mixed thoroughly as follows, shake at 1800 rpm for 2 minutes and Pipette up and down. Incubated at room temperature for 5 minutes then centrifuge at 280 g for 1 minute. Placed on a magnetic stand and wait until the liquid is clear (~8 minutes). Removed and discarded all supernatant from each well. Washed 2 times as follows. Added 200 l freshly prepared 80% EtOH to each well. Incubated on the magnetic stand for 30 seconds. Removed and discarded all supernatant from each well. Used a 20 l pipette to remove residual EtOH from each well. Air-dry on the magnetic stand for 5 minutes. Added 62.5 l RSB to each well. Removed from the magnetic stand, and then mix thoroughly as follows. Shake at 1800 rpm for 2 minutes. Pipette up and down. Incubated at room temperature for 2 minutes. Centrifuged at 280 g for 1 minute. Placed on a magnetic stand and wait until the liquid is clear (25 minutes). Transferred 60 l supernatant to the corresponding well of the IMP plate Repair Ends and Select Library Size Convert Overhangs Centrifuged ERP2 or ERP3 at 600 g for 5 seconds. Added 40 l ERP2 or ERP3 to each well, and then mixed thoroughly as follows. Shake at 1800 rpm for 2 minutes. Pipetted up and down. Centrifuged at 280 g for 1 minute. Incubated as follows, placed on the 30C microheating system with the heated lid closed for 30 minutes, and then placed on ice. Placed on the thermal cycler and run the ERP program. Each well contained 100 l. Remove Large DNA Fragments Vortexed the SPB until well-dispersed. The SPB was diluted with PCR grade water to 160 l per 100 l of end-repaired sample. The volumes were determined using the following formulas, which include 15% excess for multiple samples. Vortexed diluted SPB until well-dispersed. Added 160 l diluted SPB to each well, and then mix thoroughly as follows. Shake at 1800 rpm for 2 minutes. Pipetted up and down. Incubated at room temperature for 5 minutes. Centrifuged at 280 g for 1 minute. Placed on a magnetic stand and wait until the liquid is clear (~5 minutes). Transferred 250 l supernatant to the corresponding well of the CEP plate. Discarded remaining diluted SPB. Remove Small DNA Fragments Vortexed undiluted SPB until well-dispersed. Added 30 l undiluted SPB to each well, and then mixed thoroughly as follows. Shake at 1800 rpm for 2 minutes. Pipetted up and down. Incubated at room temperature for 5 minutes. Centrifuged at 280 g for 1 minute. Placed on a magnetic stand and wait until the liquid is clear (~5 minutes). Removed and discarded all supernatant from each well. Washed 2 times as follows. Added 200 l freshly prepared 80% EtOH to each well and Incubated on the magnetic stand for 30 seconds the Removed and discarded all supernatant from each well. Used a 20 l pipette to remove residual EtOH from each well. Air-dried on the magnetic stand for 5 minutes. Added 20 l RSB to each well. Removed from the magnetic stand, and then mixed thoroughly as follows, shake at 1800 rpm for 2 minutes and pipetted up and down. Incubated at room temperature for 2 minutes. Centrifuged at 280 g for 1 minute. Placed on a magnetic stand and wait until the liquid is clear (~5 minutes). Transferred 17.5 l supernatant to the corresponding well of the ALP plate. Adenylate 3 Ends Centrifuged the ATL or ATL2 at 600 g for 5 seconds. Added 12.5 l of ATL or ATL2 to each well, and then mixed thoroughly as follows, shake at 1800 rpm for 2 minutes and Pipetted up and down. Centrifuged at 280 g for 1 minute. Incubated as follows, placed on the 37C microheating system with the lid closed for 30 minutes. Moved to the 70C microheating system with the lid closed for 5 minutes. Placed on ice for 5 minutes. Placed on the thermal cycler and run the ATAIL70 program. Each well contains 30 l. Centrifuged at 280 g for 1 minute Ligate Adapters Removed the tape sealed from the DAP. Centrifuged the DNA adapters as follows, Prepared the DAP as follows, a Removed the plastic cover. Applied the DAP barcode. Remove LIG2 from -25C to -15C storage. Added the following reagents in the order listed to each well, and then mixed thoroughly as follows, reagent Volume (l) RSB 2.5 LIG2 2.5 DNA adapters 2.5. Shake at 1800 rpm for 2 minutes. Pipette up and down. Centrifuged at 280 g for 1 minute. Incubated as follows, placed on the 30C microheating system with the lid closed for 10 minutes, and then placed on ice. placed on the thermal cycler and run the LIG program. Each well contains 37.5 l. Centrifuged the STL at 600 g for 5 seconds. Added 5 l STL to each well, and then mixed thoroughly as follows, shake at 1800 rpm for 2 minutes. Pipetted up and down. Centrifuged at 280 g for 1 minute. Enrich DNA Fragments Amplify DNA Fragments Placed on ice and added 5 l PPC to each well. Added 20 l EPM to each well, and then mixed thoroughly as follows, shake at 1600 rpm for 20 seconds. Pipetted up and down. Centrifuged at 280 g for 1 minute. Placed on the thermal cycler and run the PCRNano program. Each well contains 50 l. Cleaned Up Amplified DNA 1 Centrifuge at 280 g for 1 minute. Vortexed SPB until well-dispersed. Added the SPB to each well. Adapter Type Volume SPB Adapter tubes 50 l DAP 47.5 l 4 Mixed thoroughly, as follows, shake at 1800 rpm for 2 minutes. pipetted up and down. Incubated at room temperature for 5 minutes. Centrifuged at 280 g for 1 minute. Placed on a magnetic stand and wait until the liquid is clear (25 minutes). Removed and discarded all supernatant from each well. Wash 2 times as follows. Added 200 l freshly prepared 80% EtOH to each well. Incubated on the magnetic stand for 30 seconds. removed and discarded all supernatant from each well. Use a 20 l pipette to remove residual EtOH from each well. Enrich DNA Fragments TruSeq Nano DNA Library Prep Reference Guide 23 11 Air-dry on the magnetic stand for 5 minutes. Added 32.5 l RSB to each well. Removed from the magnetic stand, and then mixed thoroughly as follows. Shake at 1800 rpm for 2 minutes. Pipette up and down. Incubate at room temperature for 2 minutes. Centrifuge at 280 g for 1 minute. 16 Place on a magnetic stand and wait until the liquid is clear (25 minutes). Transferred 30 l supernatant to the corresponding well of the TSP1 plate.
Submitted by: Univeristy of Pretoria and National Health Laboratory Services
Study: Characterisation of pyrazinamide resistance mechanisms in Mycobacterium tuberculosis isolates
show Abstracthide Abstract
Fifty Mycobacterium tuberculosis isolates obtained from the sputum of patients infected with tuberculosis were analysed phenotypically and genomically to characterise their pyrazinamide resistance mechanisms.Quantitative PCR and efflux inhibitors were used to determine the role of efflux in pyrazinamide resistance in this enigmatic pathogen.
Sample: Sample 13
SAMN16130549 • SRS7423724 • All experiments • All runs
Library:
Name: ARC13
Instrument: Illumina HiSeq 2500
Strategy: WGS
Source: GENOMIC
Selection: PCR
Layout: PAIRED
Runs: 1 run, 793,196 spots, 198.3M bases, 76.6Mb
Run# of Spots# of BasesSizePublished
SRR12708375793,196198.3M76.6Mb2020-09-24

ID:
11966997

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