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The genome-wide screen Tn-seq (van Opijnen et al., 2009) is very valuable tools to identify bacterial genes with a conditionally essential function, for instance genes involved in bacterial virulence. These techniques are based on the generation of a random mutant library, which is grown in a control of challenge situation (Figure 1). The advantage of using a mariner transposon for the generation of a random transposon mutant library is its insertion into TA sites, which makes the insertion in the genome highly random. In addition, an MmeI restriction site can be introduced in the inverted repeat of the transposon, without affecting the recognition by HimarC9 transposase.

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Generation and Screening of a Non-typeable Haemophilus influenzae Tn-seq Mutant Library
非分型流感嗜血杆菌Tn序列突变体库的构建和筛选

微生物学 > 微生物遗传学 > 诱/突变
作者: Jeroen D. Langereis
Jeroen D. LangereisAffiliation: Laboratory of Pediatric Infectious Diseases, Department of Pediatrics and Laboratory of Medical Immunology, Department of Laboratory Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands
For correspondence: jeroen.langereis@radboudumc.nl
Bio-protocol author page: a1207
Vol 4, Iss 5, 3/5/2014, 3601 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1065

[Abstract] The genome-wide screen Tn-seq (van Opijnen et al., 2009) is very valuable tools to identify bacterial genes with a conditionally essential function, for instance genes involved in bacterial virulence. These techniques are based on the generation of a random mutant library, which is grown in a control of challenge situation (Figure 1). The advantage of using a mariner transposon for the generation of a random transposon mutant library is its insertion into TA sites, which makes the insertion in the genome highly random. In addition, an MmeI restriction site can be introduced in the inverted repeat of the transposon, without affecting the recognition by HimarC9 transposase.

Keywords: Haemophilus influenzae(流感嗜血杆菌), Transposon sequencing(转座子序列), Tn-seq(TN seq)

[Abstract]



Figure 1. Schematic representation of the non-typeable Haemophilus influenzae tn-seq mutant library screen for survival and growth in environmental air

Materials and Reagents

  1. 1 U/μl Calf Intestinal Alkaline Phoshatase (CIAP) (New England Biolabs, catalog number: M0290S )
  2. Chloroform: isoamyl alcohol
  3. Phenol: chloroform: isoamayl alcohol
  4. Milli-Q water
  5. 10 mM dNTP mix (New England Biolabs, catalog number: N0447S )
  6. 1 mM dNTP mix
  7. Absolute ethanol
  8. 70 % Ethanol
  9. 10 mg/ml Glycogen
  10. 2 U/μl MmeI restriction enzyme (New England Biolabs, catalog number: R0637S )
  11. 10x NEBuffer 4
  12. 32 mM S-denosylmethionine
  13. 3 M NaAc (pH 5.3)
  14. 5 M NaCl
  15. 2 U/μl Phusion DNA polymerase (New England Biolabs, catalog number: M0530S )
  16. 5x Phusion HF buffer
  17. 10 U/μl T4 DNA ligase (New England Biolabs, catalog number: M0202S )
  18. 20 U/μl T4 DNA ligase
  19. 10x T4 DNA ligase buffer
  20. 2.5 U/μl T4 DNA polymerase (New England Biolabs, catalog number: M0203S )
  21. 10x T4 DNA polymerase buffer
  22. T4 polynucleotide kinase (3' phosphatase minus) (New England Biolabs, catalog number: M0236S )
  23. 100x TE buffer
  24. 1 M NaOH
  25. 50 % Glycerol
  26. 1 mM DTT
  27. 5 M NaCl
  28. 1 M MgCl2
  29. 10 mg/ml BSA
  30. 5 U/ml E.coli DNA ligase (New England Biolabs, catalog number: M0205S )
  31. 10x E.coli DNA ligase buffer
  32. 1 M Hepes (pH 7.9)
  33. HimarC9 transposase
  34. M-IV medium (Herriott et al., 1970)
  35. 1 mg/ml Hemin (Sigma-Aldrich, catalog number: H9039 )
  36. 10 mg/ml Nicotinamide adenine dinucleotide (NAD) (Sigma-Aldrich, catalog number: N7004 )
  37. Brain heart infusion medium (BHI) (BD Biosciences, catalog number: 237500 )
  38. Supplemented BHI, BHI medium containing 10 μg/ml Hemin and 2 μg/ml NAD
  39. Bacto-agar (BD Biosciences, catalog number: 212030 )
  40. Supplemented BHI plates, sBHI containing 1.5% bacto agar
  41. PBS
  42. 100 mg/ml RNase A (Roche Diagnostics, catalog number: 10109142001 )
  43. Qiagen Genomic-tip 20/G columns (QIAGEN, catalog number: 10223 )
  44. Qiagen Genomic DNA buffer set (QIAGEN, catalog number: 19060 )
  45. Minelute Reaction Cleanup Kit (QIAGEN, catalog number: 28204 )
  46. Qubit dsDNA BR assay (Life technologies, catalog number: Q32850 )
  47. Acceptor DNA
    Any type of DNA can serve as acceptor for in vitro mariner transposition. The most common types of acceptor DNA are: Chromosomal DNA of the target strain (High quality DNA is required, preferably isolated with Qiagen Genomic Tip columns) or PCR products of target genes or regions.
  48. Donor DNA
    Any type of DNA that carries a mariner transposon with MmeI restriction site in the inverted repeat can serve as donor for transposon in the in vitro mariner transposition reaction. Used pGSF8 plasmid, carrying transposon with spectinomycin resistance cassette, suitable for GAF and TnSeq (Langereis et al., 2013).
  49. Primers used for sequence adapters ligation and PCR amplification (see Appendixes)

Equipment

  1. Pipet tips : 0.5-10 μl, 2-20 μl, 20-200 μl 100-1000 μl
  2. 15 cm dishes
  3. Heating block for incubations ranging from 16 °C and 75 °C (Grant QBD2)
  4. Microcentrifuge for 1.5 ml tubes (Eppendorf, model: 5417R )
  5. Centrifuge for 50 ml tubes (Eppendorf, model: 5810 )
  6. T100 thermal cycler (Bio-Rad Laboratories)
  7. Nanodrop spectrophotometer (Thermo Fisher Scientific, Nanodrop, model: ND1000 )
  8. Incubator with 5% CO2 (BINDER GmbH, model: CB 150 )
  9. Qubit Fluorometer (Life Technologies)
  10. Bioanalyser (Agilent Technologies)

Procedure

This detailed protocol is divided into four sections:

Part I. Generation of mutant library

We provide a detailed protocol for the generation of a mutant library in non-typeable Haemophilus influenzae, but this can be used for all bacteria that are naturally competent.

Part II. Mutant library screen

As example, we provide a detailed protocol for the identification of non-typeable Haemophilus genes essential for survival in environmental air, as published before (Langereis et al., 2013).

Part III. Mutant library readout
Part IV. Data analysis

For data analysis, the web-based analysis software ESSENTIALS was used (Zomer et al., 2012). A detailed manual can be found on the website (http://bamics2.cmbi.ru.nl/websoftware/essentials/essentials_start.php).


Part I. Generation of mutant library

  1. Transposition reaction
    1. Prepare the 6x transposition buffer enough for 30 reactions fresh by combining      
      60 μl 50 % glycerol
      0.6 μl 1 M DTT
      7.5 μl Hepes (pH 7.9)
      7.5 μl 10 mg/ml BSA    
      6.0 μl 5 M NaCl
      3.0 μl 1 M MgCl2
      15.4 μl sterile dH2O
    2. Combine in a 1.5 ml tube
      3.3 μl 6x Transposition Buffer
      0.5-1.0 μg recipient DNA
      0.5-1.0 μg donor for mariner transposon
      1 μl recombinant Himar1 transposase
      Sterile dH2O until Vtotal= 20 μl
      Mix and incubate for about 4 h at 30 °C in a heating block.
    3. Inactivate transposase for 10 min at 75 °C in a heating block.
    4. Add to inactivated transposition reaction
      2 μl 3 M Sodium Acetate (pH ~5.3)
      0.5 μl 20 mg/ml glycogen
      50 μl 100% ethanol
      Mix and place in -20 °C freezer for at least 30 min.
    5. Centrifuge at maximum speed in a precooled (4 °C) microcentrifuge for 15 min.
    6. Remove supernatant with 1 ml pipet (do not touch the pellet).
      Note: At this moment it is not necessary to carefully remove al liquid.
    7. Add 250 μl of 70% ethanol (just add, do not try to resuspend the pellet).
    8. Centrifuge at maximum speed in a precooled (4 °C) microcentrifuge for 5 min.
    9. Carefully remove all supernatant with a 1 ml pipet (do not touch the pellet).
      (Optional: Centrifuge in a microcentrifuge for a few seconds to collect the remainder of the supernatant at the bottom of the tube and carefully remove all supernatant with a 200 μl pipet.)
    10. Dry the pellet on air (pellets turns from opaque to white in ~30 min).
      (Optional: Place the tubes in a heating block at 30 °C to speed up evaporation of liquid.)
    11. Dissolve pellet in 15.8 μl sterile dH2O.
  2. Repair of the transposition reaction
    1. Add to the dissolved pellet
      2 μl 10x T4 DNA polymerase Reaction Buffer
      0.2 μl 10 mg/ml BSA
      1 μl 1 mM dNTP mix      
      1 μl 2.5 U/μl T4 DNA polymerase
      Incubate for 30 min at 16 °C.
    2. Inactivate polymerase for 10 min at 75 °C in a heating block.
    3. Add to inactivated polymerase reaction
      2 μl 3 M Sodium acetate
      0.5 μl 20 mg/ml glycogen
      50 μl 100% ethanol
      Mix and incubate in -20 °C freezer for at least 30 min.
    4. Centrifuge at maximum speed in a precooled (4 °C) microcentrifuge for 15 min.
    5. Remove supernatant with 1 ml pipet (do not touch the pellet).
      Note: At this moment it is not necessary to carefully remove al liquid.
    6. Add 250 µl of 70% ethanol.
    7. Centrifuge at maximum speed in a precooled (4 °C) microcentrifuge for 5 min.
    8. Carefully remove all supernatant with a 1 ml pipet (do not touch the pellet).
      (Optional: Centrifuge in a microcentrifuge for a few seconds to collect the remainder of the supernatant at the  bottom of the tube and carefully remove all supernatant with a 200 μl pipet.)
    9. Dry the pellet on air (pellets turns from opaque to bright white in ~30 min).
      (Optional: place the tubes in a heating block at 30 °C to speed up evaporation of liquid)
    10. Dissolve pellet in 17.8 µl sterile dH2O. 
    11. Add to the dissolved pellet
      2 μl 10x E.coli DNA ligase Reaction Buffer     
      0.2 μl E.coli DNA ligase (5 U/µl)
      Incubate overnight at 16 °C.
    12. Store mutagenized DNA at -20 °C.
  3. Transformation
    1. Grow 10 ml non-typeable Haemophilus influenzae (NTHi) in BHI medium containing 10 μg/ml hemin and 2 μg/ml NAD shaking with 225 rpm at 37 °C to an OD620 of 0.3.
    2. Centrifuge the bacteria 10 min with 3,000 x g and resuspend in 10 ml PBS.
    3. Centrifuge the bacteria 10 min with 3,000 x g and resuspend in 10 ml M-IV medium and incubate 100 min shaking with 100 rpm at 37 °C.
    4. Centrifuge the bacteria 10 min with 3,000 x g and resuspend in 1 ml M-IV medium and transfer to a 1.5 ml tube.
    5. Add 1-5 μg mutagenized DNA and incubate 60 min with 100 rpm at 37 °C.
    6. Plate 1 to 100 μl per sBHI plate for over night growth at 37 °C + 5% CO2.
    7. Collect the colonies by adding 5 ml PBS + 15% glycerol on the plates and store 1 ml aliquots at -80 °C.
      Note: The number of colonies (transposon mutants) is dependent on the transformation efficiency of the NTHi strain used in this experiment. It is recommended to use a highly competent NTHi strain in order to obtain large mutant libraries. Alternatively, multiple transformations can be pooled to obtain sufficient transposon mutants, typically 10-20-fold the number of genes in the genome.

Part II. Mutant library screen

The mutant library can be used in any control and stress condition. As proof of principle, we have used growth in air enriched with 5% CO2 (control condition) or ambient air with 0.04% CO2 (stress condition). To do so, the mutant library was constructed with 5% CO2 enriched M-IV medium (M-IV medium incubated at least 2 h at 5% CO2 in an open 50 ml tube) without shaking to prevent loss of mutants while making the mutant library (generation mutant library steps 27-28).

  1. Thaw 1 ml aliquot of the NTHi mutant library at RT.
  2. Centrifuge 2 min at 10,000 x g in microcentrifuge.
  3. Remove the medium containing glycerol and resuspend the bacteria in 1 ml sBHI medium.
  4. Grow the mutant library in 5 ml sBHI medium enriched with 5% CO2 (incubate BHI medium overnight in the incubator + 5% CO2 and add hemin and NAD fresh before use) without shaking to OD620 = 0.5 and store three 1 ml aliquots with 15% glycerol at -80 °C. (Start culture)
  5. Dilute the start culture 1:100 in 5 ml 5% enriched sBHI medium (control) or sBHI medium (stress) and grow to OD620 = 0.5 with 5% CO2 (control) or ambient air (stress) at 37 °C and store three 1 ml aliquots with 15% glycerol at -80 °C for storage and start culture for next round. (Round 1)
  6. Dilute the first round culture 1:100 in 5 ml 5% enriched sBHI medium (control) or sBHI medium (stress) and grow to OD620 = 0.5 with 5% CO2 (control) or ambient air (stress) at 37 °C and store three 1 ml aliquots with 15% glycerol at -80 °C for storage and start culture for next round. (Round 2)
  7. Dilute the second round culture 1:100 in 5 ml 5% enriched sBHI medium (control) or sBHI medium (stress) and grow to OD620 = 0.5 with 5% CO2 (control) or ambient air (stress) at 37 °C and store three 1 ml aliquots with 15% glycerol at -80 °C for storage. (Round 3)
  8. Thaw the challenged mutant library at RT, centrifuge 3 min 10.000 x g and resuspend the bacteria in 1 ml buffer B1 supplemented with 2 μl RNase A solution (100 mg/ml).

Part III. Mutant library readout

  1. Mutant library chromosomal DNA isolation and digestion
    1. Thaw the challenged mutant library at RT, centrifuge 3 min 10,000 x g and resuspend the bacteria in 1 ml buffer B1 supplemented with 2 μl RNase A solution (100 mg/ml).
    2. Isolate the chromosomal DNA from the challenged mutant libraries with Qiagen Genomic Tip columns.
      1. Add 20 μl lysozyme (100 mg/ml) and 45 μl proteinase K (10 mg/ml) and incubate 30 min at 37 °C.
      2. Add 350 μl buffer B2 and incubate 30 min at 50 °C.
      3. Place a Qiagen genomic tip 20/G column on a 15 ml tube and the column with 2 ml buffer QBT.
      4. Vortex the sample and apply it to the equilibrated column.
      5. Wash the column 3x with 1 ml buffer QC.
      6. Replace the 15 ml tube and elute the DNA with 2x 1 ml buffer QF.
      7. Transfer 3x 650 μl buffer to a 1.5 ml tube, add 455 μl RT isopropanol and centrifuge immediately 15 min. with max. speed at 4 °C.
      8. Remove the isopropanol and wash with 1 ml cold 70% ethanol. Vortex briefly and centrifuge 10 min. with max. speed at 4 °C.
      9. Remove the ethanol and wash a second time with 1 ml cold 70% ethanol. Vortex briefly and centrifuge 10 min. with max. speed at 4 °C.
      10. Remove the ethanol and let the pellet dry. Do not completely dry the pellet.
      11. Resuspend the DNA pellet in 100 µl TE.
        (Optional: Incubate at 50 °C to dissolve the DNA pellet.)
    3. Prepare reaction mixture in 1.5 ml microfuge tube
      2 μg chromosomal DNA of mutant library
      5 μl 2 U/μl MmeI (=10 U)
      20 μl 10x NEBuffer 4
      0.3 μl 32 mM S-adenosylmethionine
      Vtotal with dH2O= 200 μl
      Incubate at 37 °C, >4 h.
    4. Add 1 μl 1 U/μl CIAP and mix (=1 U) and incubate at 50 °C, 30 min.
    5. Add 200 μl phenol:chloroform:isoamayl alcohol and vortex 10 sec.
    6. Centrifuge max speed, 5 min at RT.
    7. Transfer upper layer (~200 μl) to new 1.5 ml microfuge tube with 200 μl chloroform: isoamyl alcohol and vortex 10 sec.
    8. Centrifuge max speed, 5 min at RT.
    9. Transfer upper layer (~200 μl) to new 1.5 ml microfuge tube.
    10. Add to the tube
      20 μl 3 M Sodium Acetate
      0.5 μl 20 mg/ml glycogen
      500 μl 100% ethanol
      Mix and incubate in -20 °C freezer for at least 30 min.
    11. Centrifuge at maximum speed in a precooled (4 °C) microcentrifuge for 15 min.
    12. Remove supernatant with 1 ml pipet (do not touch the pellet).
      Note: At this moment it is not necessary to carefully remove al liquid.
    13. Add 500 µl of 70% ethanol.
    14. Centrifuge at maximum speed in a precooled (4 °C) microcentrifuge for 5 min.
    15. Carefully remove all supernatant with a 1 ml pipet (do not touch the pellet).
      (Optional: Centrifuge in a microcentrifuge for a few seconds to collect the remainder of the supernatant at the bottom of the tube and carefully remove all supernatant with a 200 μl pipet.)
    16. Dry the pellet on air (pellets turns from opaque to bright white in ~30 min).
      (Optional: Place the tubes in a heating block at 30 °C to speed up evaporation of liquid.) 
    17. Dissolve pellet in 20 μl dH2O.
    18. Measure the DNA concentration.
  2. Adapter annealing
    For each adapter, a F- and R-primer must be annealed (see primers listed in the appendix). A total of 12 adapters are listed in the appendix, but the number needed for the experiment (e.g. 4 or 8) can be annealed in parallel in separate tubes.
    1. For primer annealing, prepare mix in 1.5 ml microfuge tube
      5 μl 1 nmol/μl F primer
      5 μl 1 nmol/μl R primer
      0.5 μl 100x TE
      0.5 μl 5 M NaCl
      39 μl dH2O
      Incubate for 10 min at 95 °C in heating block.
    2. Remove metal tube holder from heating block and allow to cool slowly on bench to T< 30 °C.
    3. Store samples at -20 °C until further use (annealed adapters can be stored > 1 year).
    4. For 5'-phosphorylation of the annealed adapters, prepare mix in 1.5 ml microfuge tube
      2 μl annealed adapter (100 pmol/μl)
      2 μl 10x T4 DNA ligase buffer
      0.5 μl 10 U/μl T4 polynucleotide kinase (3' phosphatase minus)
      15.5 μl dH2O
    5. Incubate for 5 min at 37 °C.
    6. Incubate 10 min at 70 °C in a heat block.
    7. Remove metal tube holder from heating block and allow to cool slowly on bench to T< 30 °C.
  3. Adapter ligation and PCR amplification
    1. MmeI restriction fragments and annealed adapters were ligated in the following reaction mixture
      100 ng dephosphorylated MmeI restriction fragments
      0.2 μl (10 pmol/μl) freshly phosphorylated annealed adapter
      2 μl 10x T4 DNA ligase buffer
      0.2 μl (10 U/μl) T4 DNA ligase
      Vtotal with dH2O= 20 μl
      Incubate for over night at 16 °C.
    2. Ligated adapter and restriction fragments were PCR amplified in the following reaction mixture
      26 μl dH2O
      10 μl 5x Phusion HF buffer
      1 μl 10 mM dNTP mix
      5 μl (4 pmol/μl) PBGSF23 primer
      5 μl (4 pmol/μl) PBGSF31 primer
      2.5 μl ligation mixture
      0.5 μl Phusion DNA polymerase
      Incubate reaction according to the following PCR program
      72 °C    - 1 min
      98 °C    - 30 sec
      98 °C    - 10 sec    |
      57 °C    - 30 sec    | 25x
      72 °C    - 10 sec    |
      72 °C    - 5 min
    3. Check 2 μl of the PCR reaction on a 2.5 % agarose gel with a 100 bp ladder.
      Note: PCR product should be a single band of ~125 bp.
    4. Cleanup PCR reaction with Minelute Reaction Cleanup Kit.
      1. Mix 50 μl PCR reaction with 50 μl dH2O.
      2. Add 300 μl ERC buffer.
      3. Apply to MinElute column in 2 ml tube; centrifuge 1 min at 18,000 rpm.
      4. Discard flow-through, reuse 2 ml tube.
      5. Add 750 μl buffer PE; centrifuge 1 min at 18,000 rpm.
      6. Discard flow-through, reuse 2 ml tube.
      7. Centrifuge 1 min at maximum speed to completely dry membrane.
      8. Place MinElute column in RNase-free 1.5 ml tube.
      9. Pipet 10 μl dH2O on center of the column membrane; wait 1 min.
      10. Centrifuge 1 min at maximum speed.
    5. Measure DNA concentration with Nanodrop using d H2O as blank.
    6. Combine equimolar amounts of differently barcoded DNA probes in one tube.
    7. For quality control, perform a Qubit DNA concentration measurement and a bioanalyser run.
    8. 9 fmol of DNA probe was loaded on a Genome Analyzer II (Illumina) for sequence analysis according to the manufacturer's protocols, using a Genomic DNA Sequencing Primer (Illumina) and 36 sequencing cycles.
      For further information about Illumina sequencing see Zomer et al. (2012).

Part. IV. Data analysis

Data analysis is in detail described in Langereis et al. (2013).

  1. Generate FASTQ files with 35 bp sequences.
    Note: The first nucleotide of the Genome Analyzer II (Illumina) 36-bp sequence reads often has a poor quality and is therefore omitted.
  2. Generate a config file as Table 1 below.

    Table 1. Example for config file used for data analysis
    link
    barcode   
    transposon
    sample type
    library
    sample format
    compression
    http://bamicsb.cmbi.ru.nl/tnseq/co2_Hinfluenza/s_1_export.txt
    TCACG
    ACAGGTTGGATGAT
    target
    lib1
    export
    none
    http://bamicsb.cmbi.ru.nl/tnseq/co2_Hinfluenza/s_1_export.txt
    GATGT
    ACAGGTTGGATGAT
    control
    lib1
    export   
    none
    http://bamicsb.cmbi.ru.nl/tnseq/co2_Hinfluenza/s_1_export.txt
    TAGGC
    ACAGGTTGGATGAT
    target
    lib1
    export
    none
    http://bamicsb.cmbi.ru.nl/tnseq/co2_Hinfluenza/s_2_export.txt
    GACCA
    ACAGGTTGGATGAT
    control
    lib1
    export
    none
    http://bamicsb.cmbi.ru.nl/tnseq/co2_Hinfluenza/s_2_export.txt
    GATGT
    ACAGGTTGGATGAT
    target
    lib1
    export
    none
    http://bamicsb.cmbi.ru.nl/tnseq/co2_Hinfluenza/s_2_export.txt
    TCACG
    ACAGGTTGGATGAT
    control
    lib1
    export
    none
    http://bamicsb.cmbi.ru.nl/tnseq/co2_Hinfluenza/s_2_export.txt
    GACCA
    ACAGGTTGGATGAT
    target
    lib1
    export
    none
    http://bamicsb.cmbi.ru.nl/tnseq/co2_Hinfluenza/s_2_export.txt
    TAGGC
    ACAGGTTGGATGAT
    control
    lib1
    export
    none

  3. Choose the finished genome or upload a genbank file for the pathogen used in the screen.
  4. Upload the config .txt file.
  5. Press next.
  6. Analysis is performed with the following parameters (see Figure 2).
    1. Select TA for selected for mariner transposon mutant libraries.
    2. Select 30,000 for “library size”.
    3. Select yes for “perform repeat filtering”.
    4. Select 2 for “barcode mismatch”.
    5. Select 17 for “genomic sequence remaining of read”.
    6. Select bol for “barcode side”.
    7. Select eol for “transposon inverted repeat side”.
    8. Select 14 for “minimal sequence match”.
    9. Select 1(=forward) for “strand to align”.
    10. Select truncated.ptt for “3’ truncated genes for matching insertion site”.
    11. Select yes for “remove genomic position bias”.
    12. Select TMM for “normalization”.
    13. Select qCML for “paired analysis”.
    14. Select tagwise for “modeling of variance”.
    15. Select 10 for “amount of smoothing”.
    16. Select BH for “p-value adjustment method”.
    17. Select corrected for “p-value”.
    18. Select 20 for “minimal number of reads”.
    19. Select yes for “create ZIP archive”.
  7. Press proceed
    Note: Analysis can take up to a few hours.
  8. Unzip the created zip file and the file gene_alloutputmerged.tsv contains the data analysis for conditionally essential genes.


    Figure 2. Screenshot of the analysis parameters on the ESSENTIALS website

Acknowledgments

This protocol is adapted from a previously published paper: Langereis et al. (2013).

References

  1. Langereis, J. D., Zomer, A., Stunnenberg, H. G., Burghout, P. and Hermans, P. W. (2013). Nontypeable Haemophilus influenzae carbonic anhydrase is important for environmental and intracellular survival. J Bacteriol 195(12): 2737-2746.
  2. Herriott, R. M., Meyer, E. M. and Vogt, M. (1970). Defined nongrowth media for stage II development of competence in Haemophilus influenzae. J Bacteriol 101(2): 517-524.
  3. Illumina sequencing technology. http://res.illumina.com/documents/products/techspotlights/techspotlight_sequencing.pdf.
  4. van Opijnen, T., Bodi, K. L. and Camilli, A. (2009). Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6(10): 767-772. 
  5. Zomer, A., Burghout, P., Bootsma, H. J., Hermans, P. W. and van Hijum, S. A. (2012). ESSENTIALS: software for rapid analysis of high throughput transposon insertion sequencing data. PLoS One 7(8): e43012.

Appendixes

Table 2. Primers used for tn-seq analysis*

Characteristics
sequence (5'-3')
Adapter
F primer with ATCACG barcode
TTCCCTACACGACGCTCTTCCGATCTATCACGNN
A
R primer with ATCACG barcode
P-CGTGATAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F primer with CGATGT barcode
TTCCCTACACGACGCTCTTCCGATCTCGATGTNN
B
R primer with CGATGT barcode
P-ACATCGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F primer with TTAGGC barcode
TTCCCTACACGACGCTCTTCCGATCTTTAGGCNN
C
R primer with TTAGGC barcode
TTCCCTACACGACGCTCTTCCGATCTTGACCANN
F primer with TGACCA barcode
TTCCCTACACGACGCTCTTCCGATCTTGACCANN
D
R primer with TGACCA barcode
P-TGGTCAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F primer with ACAGTG barcode
TTCCCTACACGACGCTCTTCCGATCTACAGTGNN
E
R primer with ACAGTG barcode
P-CACTGTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F primer with GCCAAT barcode
TTCCCTACACGACGCTCTTCCGATCTGCCAATNN
F
R primer with GCCAAT barcode
P-ATTGGCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F primer with CAGATC barcode
TTCCCTACACGACGCTCTTCCGATCTCAGATCNN
G
R primer with CAGATC barcode
P-GATCTGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F primer with ACTTGA barcode
TTCCCTACACGACGCTCTTCCGATCTACTTGANN
H
R primer with ACTTGA barcode   
P-TCAAGTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F primer with GATCAG barcode
TTCCCTACACGACGCTCTTCCGATCTGATCAGNN
I
R primer with GATCAG barcode
P-CTGATCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F primer with TAGCTT barcode
TTCCCTACACGACGCTCTTCCGATCTTAGCTTNN
J
R primer with TAGCTT barcode
P-AAGCTAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F primer with GGCTAC barcode
TTCCCTACACGACGCTCTTCCGATCTGGCTACNN
K
R primer with GGCTAC barcode
P-GTAGCCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F primer with CTTGTA barcode
TTCCCTACACGACGCTCTTCCGATCTCTTGTANN
L
R primer with CTTGTA barcode
P-TACAAGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
Amplification primer 1   
CAAGCAGAAGACGGCATACGAAGACCGGGGACTTATCATCCAACCTGT
Amplification primer 2
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
*All primers were PAGE purified; P, phosphorylated; Barcodes are based on Illumina TruSeq Technology



图1.不可分型 流感嗜血杆菌 tn-seq突变体文库筛选在环境空气中的存活和生长的示意图< br />

材料和试剂

  1. 1U /μl小牛肠碱性磷酸酶(CIAP)(New England Biolabs,目录号:M0290S)
  2. 氯仿:异戊醇
  3. 苯酚:氯仿:异戊醇
  4. Milli-Q水
  5. 10mM dNTP混合物(New England Biolabs,目录号:N0447S)
  6. 1mM dNTP mix
  7. 绝对乙醇
  8. 70%乙醇
  9. 10 mg/ml糖原
  10. 2U /μlMmeI限制酶(New England Biolabs,目录号:R0637S)
  11. 10x NEBuffer 4
  12. 32mM S-去唾液酸甲硫氨酸
  13. 3 M NaAc(pH 5.3)
  14. 5 M NaCl
  15. 2U /μlPhusion DNA聚合酶(New England Biolabs,目录号:M0530S)
  16. 5x Phusion HF缓冲液
  17. 10U /μlT4 DNA连接酶(New England Biolabs,目录号:M0202S)
  18. 20 U /μlT4 DNA连接酶
  19. 10×T4DNA连接酶缓冲液
  20. 2.5U /μlT4 DNA聚合酶(New England Biolabs,目录号:M0203S)
  21. 10x T4 DNA聚合酶缓冲液
  22. T4多核苷酸激酶(3'磷酸酶缺失)(New England Biolabs,目录号:M0236S)
  23. 100x TE缓冲区
  24. 1 M NaOH
  25. 50%甘油
  26. 1 mM DTT
  27. 5 M NaCl
  28. 1 M MgCl 2
  29. 10mg/ml BSA
  30. 5U/ml大肠杆菌DNA连接酶(New England Biolabs,目录号:M0205S)
  31. 10x大肠杆菌 DNA连接酶缓冲液
  32. 1M Hepes(pH 7.9)
  33. HimarC9转座酶
  34. M-IV培养基(Herriott等人,1970)
  35. 1mg/ml Hemin(Sigma-Aldrich,目录号:H9039)
  36. 10mg/ml烟酰胺腺嘌呤二核苷酸(NAD)(Sigma-Aldrich,目录号:N7004)
  37. 脑心浸液介质(BHI)(BD Biosciences,目录号:237500)
  38. 补充的含有10μg/ml血红素和2μg/ml NAD的BHI,BHI培养基
  39. 细菌琼脂(BD Biosciences,目录号:212030)
  40. 补充的BHI板,含有1.5%细菌琼脂的sBHI
  41. PBS
  42. 100mg/ml RNA酶A(Roche Diagnostics,目录号:10109142001)
  43. Qiagen Genomic-tip 20/G柱(QIAGEN,目录号:10223)
  44. Qiagen基因组DNA缓冲液(QIAGEN,目录号:19060)
  45. Minelute Reaction Cleanup Kit(QIAGEN,目录号:28204)
  46. Qubit dsDNA BR测定(Life technologies,目录号:Q32850)
  47. 接受体DNA
    任何类型的DNA可以作为受体用于体外水手转座。 最常见的受体DNA类型是:靶菌株的染色体DNA(需要高质量DNA,优选用Qiagen Genomic Tip柱分离) 或靶基因或区域的PCR产物
  48. 供体DNA
    携带在反向重复中具有MmeI限制性位点的水平转座子的任何类型的DNA可以在体外水手转座反应中用作转座子的供体。 使用的pGSF8质粒,携带具有壮观霉素抗性盒的转座子,适用于GAF和TnSeq(Langereis等人,2013)。
  49. 用于序列衔接子连接和PCR扩增的引物(参见附录)

设备

  1. 吸头提示:0.5-10μl,2-20μl,20-200μl100-1000μl
  2. 15厘米的菜肴
  3. 用于温度范围为16°C至75°C(Grant QBD2)的加热块
  4. 微量离心机用于1.5ml管(Eppendorf,型号:5417R)
  5. 离心50毫升管(Eppendorf,型号:5810)
  6. T100热循环仪(Bio-Rad Laboratories)
  7. Nanodrop分光光度计(Thermo Fisher Scientific,Nanodrop,型号:ND1000)
  8. 具有5%CO 2的培养箱(BINDER GmbH,型号:CB 150)
  9. Qubit荧光计(Life Technologies)
  10. 生物分析仪(Agilent Technologies)

程序

该详细协议分为四个部分:

第一部分。突变体文库的产生

我们提供了在非典型流感嗜血杆菌中产生突变体文库的详细方案,但这可以用于所有天然感染的细菌。

第二部分。 突变库屏幕

作为实例,我们提供了用于鉴定环境空气中存活所必需的不可分型的嗜血杆菌基因的详细方案(Langereis等人,2013)。

第三部分。 突变库读数
第四部分。 数据分析

对于数据分析,使用基于网络的分析软件ESSENTIALS(Zomer等人,2012)。 详细的手册可在网站上找到( http://bamics2.cmbi.ru .nl/websoftware/essentials/essentials_start.php )。


第一部分。突变体文库的产生

  1. 转座反应
    1. 准备6x转置缓冲区足够30个反应,通过结合      
      60μl50%甘油 0.6μl1M DTT
      7.5μlHepes(pH7.7)
      7.5微升10毫克/毫升BSA   
      6.0μl5M NaCl
      3.0μl1M MgCl 2/v/v 15.4μl无菌dH 2 O
    2. 在1.5 ml管中合并
      3.3μl6x转座缓冲液
      0.5-1.0μg受体DNA 0.5-1.0μg供体用于水手转座子
      1μl重组Himar1转座酶
      无菌dH 2 O直至V sub总计20μl
      混合并在加热块中在30℃下孵育约4小时。
    3. 在加热块中于75℃灭活转座酶10分钟。
    4. 添加到失活的转座反应
      2μl3 M醋酸钠(pH〜5.3)
      0.5μl20mg/ml糖原
      50μl100%乙醇
      混合并置于-20°C冰箱至少30分钟。
    5. 在预冷(4℃)微量离心机中以最大速度离心15分钟。
    6. 用1 ml移液管除去上清液(不要接触沉淀)。
      注意:此时,无需小心移除液体。
    7. 加入250μl的70%乙醇(只是添加,不要尝试重悬颗粒)。
    8. 在预冷(4℃)微量离心机中以最大速度离心5分钟。
    9. 用1ml移液管小心取出所有上清液(不要接触沉淀)。
      (任选:在微量离心机中离心几秒钟以收集管底部的剩余上清液,并用200μl移液管小心地除去所有上清液。)
    10. 在空气中干燥颗粒(颗粒在〜30分钟内从不透明变成白色) (可选:将管放置在30℃的加热块中,以加速液体的蒸发。)
    11. 将沉淀物溶解在15.8μl无菌dH 2 O中
  2. 修复转座反应
    1. 加入溶解的颗粒
      2μl10x T4 DNA聚合酶反应缓冲液
      0.2μl10mg/ml BSA
      1μl1 mM dNTP mix      
      1μl2.5U /μlT4 DNA聚合酶
      在16℃下孵育30分钟。
    2. 灭活聚合酶在75°C在加热块中10分钟。
    3. 加入灭活的聚合酶反应物
      2μl3M乙酸钠
      0.5μl20mg/ml糖原
      50μl100%乙醇
      混合并孵育在-20°C冰箱至少30分钟。
    4. 在预冷(4℃)微量离心机中以最大速度离心15分钟。
    5. 用1 ml移液管除去上清液(不要接触沉淀)。
      注意:此时,无需小心移除液体。
    6. 加入250μl的70%乙醇。
    7. 在预冷(4℃)微量离心机中以最大速度离心5分钟。
    8. 用1ml移液管小心取出所有上清液(不要接触沉淀)。
      (任选:在微量离心机中离心几秒钟以收集管底部的剩余上清液,并用200μl移液管小心地除去所有上清液。)
    9. 将颗粒在空气中干燥(在〜30分钟内,颗粒从不透明转变为亮白色) (可选:将管放置在30°C的加热块中,以加速液体的蒸发)
    10. 将沉淀物溶解在17.8μl无菌dH 2 O中。
    11. 加入溶解的颗粒
      2μl10x大肠杆菌 DNA连接酶反应缓冲液     
      0.2μl大肠杆菌 DNA连接酶(5U /μl) 在16℃孵育过夜。
    12. 储存诱变DNA在-20°C
  3. 转型
    1. 在37℃下以225rpm在含有10μg/ml氯高铁血红素和2μg/ml NAD的BHI培养基中生长10ml不可分型流感嗜血杆菌(NTHi),直至OD <620> sub> 0.3。
    2. 用3,000×g离心细菌10分钟,并重悬于10ml PBS中。
    3. 用3,000×g离心细菌10分钟,并重悬于10ml M-IV培养基中,并在37℃下以100rpm振摇孵育100分钟。
    4. 用3,000×g离心细菌10分钟,并重悬于1ml M-IV培养基中并转移至1.5ml管中。
    5. 加入1-5μg诱变的DNA并在37℃下以100rpm孵育60分钟。
    6. 板1至100μl每sBHI板在37℃+ 5%CO 2下过夜生长。
    7. 通过在板上加入5ml PBS + 15%甘油来收集菌落,并在-80℃下存储1ml等分试样。
      注意:集落(转座子突变体)的数量取决于本实验中使用的NTHi菌株的转化效率。建议使用高度胜任的NTHi菌株,以获得大突变体文库。或者,可以合并多次转化以获得足够的转座子突变体,通常是基因组中基因数量的10-20倍。

第二部分。突变库屏幕

突变体文库可用于任何对照和胁迫条件。作为原理的证明,我们已经使用在富集有5%CO 2(对照条件)或具有0.04%CO 2(应力条件)的环境空气的空气中的生长。为此,用5%CO 2富集的M-IV培养基(M-IV培养基,在5%CO 2下培养至少2小时)构建突变体文库。开放的50ml管),不摇动以防止突变体的损失,同时制备突变体文库(生成突变体文库步骤27-28)。

  1. 解冻1毫升等分的NTHi突变体文库在室温。
  2. 在微量离心机中以10,000×g离心2分钟。
  3. 取出含有甘油的培养基,并在1ml sBHI培养基中重悬细菌。
  4. 在5mL富含5%CO 2的sBHI培养基中培养突变体文库(在培养箱+ 5%CO 2中孵育BHI培养基过夜,并在使用前加入血红素和NAD新鲜) ),不振荡至OD 620 = 0.5,并在-80℃下存储三个1ml等分试样与15%甘油。 (开始培养)
  5. 在5ml 5%富集的sBHI培养基(对照)或sBHI培养基(应激)中稀释起始培养物1:100,并用5%CO 2培养基培养至OD 620 = (对照)或环境空气(应激)在37℃,并存储三个1毫升等分试样与15%甘油在-80℃下存储和开始培养下一轮。 (第1轮)
  6. 在5ml 5%富集的sBHI培养基(对照)或sBHI培养基(应激)中稀释第一轮培养物1:100,并用5%CO 2/ml培养至OD 620 = >(对照)或环境空气(胁迫),并在-80℃下存储三个1ml含15%甘油的等分试样用于储存并开始培养用于下一轮。 (第二轮)
  7. 在5ml 5%富集的sBHI培养基(对照)或sBHI培养基(应激)中稀释第二轮培养物1:100,并用5%CO 2浓度培养至OD 620 = >(对照)或环境空气(应激)在37℃,并存储三个1毫升等分试样与15%甘油在-80℃下储存。 (第3轮)
  8. 在RT下解冻被攻击的突变体文库,离心3分钟10.000×g,并将细菌重悬在补充有2μlRNA酶A溶液(100mg/ml)的1ml缓冲液B1中。

第三部分。突变体文库读出

  1. 突变体文库染色体DNA的分离和消化
    1. 解冻挑战突变库在室温,离心3分钟10,000×g,并重悬在1毫升缓冲液B1添加2微升RNA酶A溶液(100毫克/毫升)。
    2. 用Qiagen Genomic Tip柱分离来自激发的突变体文库的染色体DNA。
      1. 加入20μl溶菌酶(100 mg/ml)和45μl蛋白酶K(10 mg/ml),并在37℃孵育30分钟。
      2. 加入350μl缓冲液B2,并在50°C孵育30分钟。
      3. 将Qiagen基因组尖端20/G柱置于15ml管上,柱子用2ml缓冲液QBT。
      4. 涡旋样品并将其应用于平衡的柱。
      5. 用1ml缓冲液QC洗涤柱3次。
      6. 更换15毫升管,用2×1毫升缓冲液QF洗脱DNA。
      7. 转移3x 650微升缓冲液到1.5毫升管,加入455微升RT异丙醇,立即离心15分钟。 最大。 速度在4℃。
      8. 除去异丙醇并用1ml冷的70%乙醇洗涤。 短暂涡旋并离心10分钟。 最大。 速度在4℃。
      9. 除去乙醇,并用1ml冷的70%乙醇洗涤第二次。 短暂涡旋并离心10分钟。 最大。 速度在4℃。
      10. 取出乙醇,让沉淀干燥。 不要完全干燥沉淀。
      11. 重悬在100微升TE的DNA沉淀。
        (可选:在50°C孵育以溶解DNA沉淀。)
    3. 在1.5ml微量离心管中制备反应混合物
      2μg突变体文库的染色体DNA 5μl2U /μlMmeI(= 10U)
      20μl10x NEBuffer 4
      0.3μl32mM S-腺苷甲硫氨酸 V total 与dH <2> O = 200微升
      在37℃,> 4小时孵育。
    4. 加入1μl1U /μlCIAP并混合(= 1U),并在50℃孵育30分钟。
    5. 加入200μl苯酚:氯仿:异戊醇,涡旋10秒。
    6. 离心最大速度,室温下5分钟。
    7. 转移上层(〜200微升)到新的1.5毫升微量离心管用200微升氯仿:异戊醇,涡旋10秒。
    8. 离心最大速度,室温下5分钟。
    9. 转移上层(〜200微升)到新的1.5毫升微量离心管。
    10. 添加到管
      20μl3 M醋酸钠
      0.5μl20mg/ml糖原
      500μl100%乙醇
      混合并孵育在-20°C冰箱至少30分钟。
    11. 在预冷(4℃)微量离心机中以最大速度离心15分钟。
    12. 用1 ml移液管除去上清液(不要接触沉淀)。
      注意:此时,无需小心移除液体。
    13. 加入500μl的70%乙醇。
    14. 在预冷(4℃)微量离心机中以最大速度离心5分钟。
    15. 用1ml移液管小心取出所有上清液(不要接触沉淀)。
      (任选:在微量离心机中离心几秒钟以收集管底部的剩余上清液,并用200μl移液管小心地除去所有上清液。)
    16. 将颗粒在空气中干燥(在〜30分钟内,颗粒从不透明转变为亮白色) (可选:将管放置在30℃的加热块中,以加速液体的蒸发。
    17. 将沉淀溶于20μldH 2 O中。
    18. 测量DNA浓度。
  2. 适配器退火
    对于每个衔接子,F-和R-引物必须退火(参见附录中列出的引物)。 附录中总共列出了12个衔接子,但是实验所需的数量(例如<4>或4)可以在单独的试管中平行退火。
    1. 对于引物退火,在1.5ml微量离心管中制备混合物
      5μl1 nmol /μlF引物
      5μl1 nmol /μlR引物
      0.5μl100x TE
      0.5μl5M NaCl
      39μldH2O
      在95℃在加热块中孵育10分钟。
    2. 从加热块移除金属管保持器,并允许在工作台上缓慢冷却至T < 30℃。
    3. 将样品储存在-20℃直到进一步使用(退火的衔接子可以储存> 1年)。
    4. 对于退火的衔接子的5'-磷酸化,在1.5ml微量离心管中制备混合物
      2μl退火适配器(100 pmol /μl)
      2μl10×T4 DNA连接酶缓冲液
      0.5μl10U /μlT4多核苷酸激酶(3'磷酸酶减去)
      15.5μldH 2 O
    5. 在37℃孵育5分钟。
    6. 在70℃在加热块中孵育10分钟。
    7. 从加热块移除金属管保持器,并允许在工作台上缓慢冷却至T < 30℃。
  3. 衔接子连接和PCR扩增
    1. 将MmeI限制性片段和退火的衔接子连接到下列反应混合物中 100ng去磷酸化的MmeI限制性片段 0.2μl(10 pmol /μl)新鲜磷酸化退火衔接子
      2μl10×T4 DNA连接酶缓冲液
      0.2μl(10U /μl)T4 DNA连接酶
      V total with dH sub 2 O = 20μl
      在16°C孵育过夜。
    2. 将连接的接头和限制性片段在下列反应混合物中进行PCR扩增 26微升dH 2 O 2 / 10μl5x Phusion HF缓冲液
      1μl10 mM dNTP mix
      5μl(4pmol /μl)PBGSF23引物
      5μl(4 pmol /μl)PBGSF31引物
      2.5μl连接混合物
      0.5μlPhusion DNA聚合酶
      根据以下PCR程序孵育反应物
      72°C    - 1分钟
      98°C    - 30秒
      98°C    - 10秒   |
      57°C    - 30秒   | 25x
      72°C    - 10秒   |
      72°C    - 5分钟
    3. 检查2μlPCR反应在2.5%琼脂糖凝胶上,100 bp阶梯 注意: PCR产物应为约125 bp的单一条带。
    4. 用Minelute反应清除试剂盒进行纯化PCR反应。
      1. 将50μlPCR反应物与50μldH 2 O混合
      2. 加入300微升ERC缓冲液。
      3. 应用于2ml管中的MinElute柱; 在18,000rpm离心1分钟。
      4. 丢弃流过,重复使用2毫升管。
      5. 加入750μl缓冲液PE; 在18,000rpm离心1分钟。
      6. 丢弃流过,重复使用2毫升管。
      7. 以最大速度离心1分钟以完全干燥膜。
      8. 将MinElute柱放置在无RNA酶的1.5ml管中。
      9. 在柱膜的中心吸移10μldH 2 O; 等待1分钟。
      10. 以最大速度离心1分钟。
    5. 使用Nanodrop测量DNA浓度,使用d H 2 O作为空白
    6. 在一个管中组合等摩尔量的不同条形码的DNA探针。
    7. 对于质量控制,执行Qubit DNA浓度测量和生物分析仪运行。
    8. 将9fmol的DNA探针装载在Genome Analyzer II(Illumina)上,根据制造商的方案使用基因组DNA测序引物(Illumina)和36个测序循环进行序列分析。
      有关Illumina测序的更多信息,请参阅 Zomer 。 (2012)。

部分。 IV。 数据分析

数据分析详细描述在Langereis等人中。 (2013年)。

  1. 生成35 bp序列的FASTQ文件。
    注意:基因组分析仪II(Illumina)的第一个核苷酸36-bp序列读取通常具有较差的质量,因此省略。
  2. 生成如下表1所示的配置文件。

    表1.用于数据分析的配置文件示例
    链接
    条形码   
    转座子
    样品类型

    示例格式
    压缩
    http://bamicsb.cmbi.ru.nl/tnseq/co2_Hinfluenza/s_1_export.txt
    TCACG
    ACAGGTTGGATGAT
    目标
    lib1
    导出

    http://bamicsb.cmbi.ru.nl/tnseq/co2_Hinfluenza/s_1_export.txt
    GATGT
    ACAGGTTGGATGAT
    控制
    lib1
    导出   

    http://bamicsb.cmbi.ru.nl/tnseq/co2_Hinfluenza/s_1_export.txt
    TAGGC
    ACAGGTTGGATGAT
    目标
    lib1
    导出

    http://bamicsb.cmbi.ru.nl/tnseq/co2_Hinfluenza/s_2_export.txt
    GACCA
    ACAGGTTGGATGAT
    控制
    lib1
    导出

    http://bamicsb.cmbi.ru.nl/tnseq/co2_Hinfluenza/s_2_export.txt
    GATGT
    ACAGGTTGGATGAT
    目标
    lib1
    导出

    http://bamicsb.cmbi.ru.nl/tnseq/co2_Hinfluenza/s_2_export.txt
    TCACG
    ACAGGTTGGATGAT
    控制
    lib1
    导出

    http://bamicsb.cmbi.ru.nl/tnseq/co2_Hinfluenza/s_2_export.txt
    GACCA
    ACAGGTTGGATGAT
    目标
    lib1
    导出

    http://bamicsb.cmbi.ru.nl/tnseq/co2_Hinfluenza/s_2_export.txt
    TAGGC
    ACAGGTTGGATGAT
    控制
    lib1
    导出


  3. 选择完成的基因组或上传用于屏幕中病原体的genbank文件。
  4. 上传config .txt文件。
  5. 按下一步。
  6. 使用以下参数进行分析(参见图2)。
    1. 选择TA为选定的水手转座子突变体库。
    2. 为"库大小"选择30,000。
    3. 对"执行重复过滤"选择是。
    4. 为"条码不匹配"选择2
    5. 为"读取的基因组序列剩余"选择17。
    6. 为"条码边"选择bol。
    7. 选择eol作为"转座子反向重复侧"。
    8. 为"最小序列匹配"选择14。
    9. 为"strand to align"选择1(= forward)。
    10. 选择truncated.ptt为"3'截断基因匹配插入位点"。
    11. 为"删除基因组位置偏差"选择"是"。
    12. 选择TMM进行"归一化"。
    13. 为"配对分析"选择qCML。
    14. 为"方差建模"选择标签方式。
    15. 为"平滑量"选择10。
    16. 为"p值调整方法"选择BH。
    17. 选择"p值"校正。
    18. 为"最小读取数"选择20。
    19. 对"创建ZIP存档"选择是。

  7. 注意:分析最多可能需要几个小时。
  8. 解压缩创建的zip文件,文件gene_alloutputmerged.tsv包含条件必需基因的数据分析。


    图2. ESSENTIALS网站上的分析参数的屏幕截图

致谢

该协议改编自以前发表的论文: Langereis (2013)。

参考文献

  1. Langereis,J.D.,Zomer,A.,Stunnenberg,H.G.,Burghout,P.and Hermans,P.W。(2013)。 不可分型的流感嗜血杆菌碳酸酐酶对环境和细胞内存活很重要。/a> J Bacteriol 195(12):2737-2746。
  2. Herriott,R.M.,Meyer,E.M。和Vogt,M。(1970)。 为流感嗜血杆菌中的II期发展定义非传染性媒介。 J Bacteriol 101(2):517-524。
  3. Illumina测序技术。 http://res.illumina.com/documents/products/techspotlights/techspotlight_sequencing.pdf 。
  4. van Opijnen,T.,Bodi,K.L.和Camilli,A。(2009)。 Tn-seq:high微生物中的适应性和遗传相互作用研究的平行测序。国家方法 6(10):767-772。
  5. Zomer,A.,Burghout,P.,Bootsma,H.J.,Hermans,P.W.and van Hijum,S.A。(2012)。 必备软件:用于快速分析高通量转座子插入测序数据的软件 PLoS One 7(8):e43012。

附录

T 能2.用于tn-seq分析的引物*

特性
序列(5'-3')
适配器
F引物与ATCACG条码
TTCCCTACACGACGCTCTTCCGATCTATCACGNN
A
R引物与ATCACG条形码
P-CGTGATAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F引物与CGATGT条码
TTCCCTACACGACGCTCTTCCGATCTCGATGTNN
B
R引物与CGATGT条码
P-ACATCGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F引物与TTAGGC条形码
TTCCCTACACGACGCTCTTCCGATCTTTAGGCNN
C
R引物与TTAGGC条形码
TTCCCTACACGACGCTCTTCCGATCTTGACCANN
F引物与TGACCA条形码
TTCCCTACACGACGCTCTTCCGATCTTGACCANN
D
R引物与TGACCA条形码
P-TGGTCAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F引物与ACAGTG条码
TTCCCTACACGACGCTCTTCCGATCTACAGTGNN
E
R引物与ACAGTG条码
P-CACTGTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F引物与GCCAAT条码
TTCCCTACACGACGCTCTTCCGATCTGCCAATNN
F
R引物与GCCAAT条码
P-ATTGGCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F引物与CAGATC条形码
TTCCCTACACGACGCTCTTCCGATCTCAGATCNN
G
R引物与CAGATC条形码
P-GATCTGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F引物与ACTTGA条形码
TTCCCTACACGACGCTCTTCCGATCTACTTGANN
H
R引物与ACTTGA条形码   
P-TCAAGTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F引物与GATCAG条形码
TTCCCTACACGACGCTCTTCCGATCTGATCAGNN
I
R引物与GATCAG条形码
P-CTGATCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F引物与TAGCTT条形码
TTCCCTACACGACGCTCTTCCGATCTTAGCTTNN
J
R引物与TAGCTT条形码
P-AAGCTAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F引物与GGCTAC条形码
TTCCCTACACGACGCTCTTCCGATCTGGCTACNN
K
R引物与GGCTAC条形码
P-GTAGCCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
F引物与CTTGTA条形码
TTCCCTACACGACGCTCTTCCGATCTCTTGTANN
L
R引物与CTTGTA条形码
P-TACAAGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-P
扩增引物1   
CAAGCAGAAGACGGCATACGAAGACCGGGGACTTATCATCCAACCTGT
扩增引物2
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
*所有引物均为PAGE纯化; 磷酸化; 条形码基于Illumina TruSeq技术

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How to cite this protocol: Langereis, J. D. (2014). Generation and Screening of a Non-typeable Haemophilus influenzae Tn-seq Mutant Library . Bio-protocol 4(5): e1065. DOI: 10.21769/BioProtoc.1065; Full Text



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