我们已经开发出全面的,无偏见的全基因组的屏幕,了解基因药物和基因与环境的相互作用。筛选这些突变集合的方法。
凭借在新一代测序技术的进步,我们几乎每天都获得新的基因组序列。这些进步的步伐正在加快,前途更大的深度和广度。鉴于这些非凡的进步,需要快速,并行的方法,以确定基因功能变得更加重要。集合的全基因组的缺失突变体, 酵母菌和大肠杆菌大肠杆菌曾担任基因功能的功能特性的工作母机,但这种方法是不可伸缩的,目前的基因缺失方法要求每个成千上万的基因组成基因组中被删除,并核实的。只有这项工作完成后,我们可以追求高通量的表型。在过去的十年,我们的实验室已提炼组合的竞争力,小型化,高通量的基因组范围内,可并行执行检测。这是可能的并行,因为DNA“标签”,或“条码”,到每个突变列入,条形码突变的代理服务和一个可以衡量条码丰评估突变体健身。在这项研究中,我们设法填补DNA序列和条形码突变集合之间的差距。而要做到这一点,我们引入一个结合转座子中断条码的方法,开辟了并行条码检测新测序,但较差的特点微生物。为了说明这种方法,我们提出一个新的白色念珠菌条形码中断收集和描述如何将两个芯片为基础的下一代测序为基础的平台,可以用来收集万- 100万在一次实验中基因-基因和基因药物的相互作用。
在这里,我们提出了一个协议,适度的修改,可以很容易地适应广泛的现有馆藏的创建标签突变馆藏的条形码不同的微生物突变集合。我们强调,虽然我们已经报道了标签的转座子突变致病性酵母菌 C.一个协议白色念珠菌 ,一个非常类似的协议,可以适应各种各样的单细胞真菌。修改,该协议运作良好13中的细菌,和目前正在建设中一些额外的真菌和细菌的基因组的集合。目前,这个实验提供了唯一的综合性,全基因组基因的小分子的相互作用的公正屏幕。其中特别引人注目的检测功能是没有的基因或小分子的先验知识是必需的。尽管这些检测的范围和权力,其可转让给其他实验室已经有点阻碍,由最初的资本投资和信息工具,分析的结果。通过结合分析的强大工具的下一代序列读数,我们期望通过增加。
The authors have nothing to disclose.
我们感谢罗恩戴维斯,亚当Deutschbauer,整个臀部在多伦多大学的讨论和意见的跳实验室。 CN是由国家人类基因组研究所(批准号HG000205),RO1 HG003317,CIHR澳门币84305,加拿大癌症协会(#020380)的赠款支持。联办是由斯坦福大学的基因组培训计划(批准数的T32 HG00044从国家人类基因组研究所)和国立卫生研究院(批准号P01 GH000205)支持。 GG是在NHGRI RO1 HG003317和加拿大癌症协会,格兰特#020380,运输署和唐纳利测序中心的支持,部分是从加拿大创新到DRS基金会的赠款支持。布兰达安德鲁斯和杰克布拉特。 AMS是由多伦多大学打开奖学金支持。
Antibiotics | Vendor and catalogue numbers |
Carbenicillin | Sigma, part# C1613 |
Kanamycin | Sigma, part# K1876 |
spectinomycin | Sigma, part# S0692 |
Chloramphenicol | Sigma, part# C0378 |
DNA Clean-up and concentration kits | |
QIAprep Spin Miniprep Kit | Qiagen, part# 27106 |
HiSpeed Plasmid Maxi Kit | Qiagen, part# 12663 |
QIAquick PCR Purification Kit | Qiagen, part# 28106 |
QIAquick Gel Extraction Kit | Qiagen, part# 28704 |
PCR and electrophoresis reagents | |
Taq DNA Polymerase (Mg-free) Buffer | New England Biolabs, part# M0320L |
Deoxynucleotide Solution Mix | New England Biolabs, part# N0447L |
25 mM MgCl2 | Sigma, part# 63036 |
Agarose, loading dye, and nucleic acid stain suitable for gel electrophoresis | Various |
10X TAE buffer | Sigma, part# T8280 |
1 Kb Plus DNA Ladder | Invitrogen, part# 10787026 |
YPD broth | |
10 g of yeast extract | Sigma, part# Y1625 |
20 g Bacto peptone | BD Biosciences, part# 211677 |
20 g of dextrose | Sigma, part# D9434 |
Labware | |
Multichannel pipettes (1000, 200, and 20 μL) | Various |
Disposable pipetting reservoirs | Various |
15 and 50 mL centrifuge tubes | Various |
96- and 384-Well Deep Well Plates | Axygen Scientific, part# P-2ML-SQ-C-S & P-384240SQCS |
96-well and 384-well PCR plates and seal film | Various |
Plate roller for sealing multi-well plates | Sigma, part# R1275 |
30°C and 37°C shaking incubators for growing bacterial and yeast on plates and in tubes | Various |
In vitro transposon mutagenesis | |
EZ-Tn5 Transposase | Epicentre Biotechnologies, part# TNP92110 |
High-throughput transformation | |
Seqprep 96 HT Plasmid Prep Kit | Edge Biosystems, part# 84359 |
polyethylene glycol, molecular weight 3350 | Various |
lithium acetate | Sigma, part# 517992 |
6-well plates, sterile | Corning, part# 3335 |
50 mg/mL uridine | Sigma, part# U3750 |
100X Tris-EDTA buffer solution | Sigma, part# T9285 |
1X TE/0.1M LiOAc | Various |
salmon testis DNA | Sigma, part# 1626 |
Growth of barcoded collections | |
48-well plates; if growing cultures in plates | Greiner, part# M9437 |
Adhesive plate seals | ABgene, part# AB-0580 |
200 mL culture flasks | Various |
Spectrophotometer capable of absorbance | Various |
Temperature-controlled shaker for 250 ml flasks or shaking spectrophotometer | Various |
Safe-Lock Microcentrifuge Tube, 2 mL | Eppendorf, part# 0030 120.094 |
Hybridization equipment | |
Hybridization Oven 640 | Affymetrix, part# 800138 |
GeneChip Fluidic Station 450 | Affymetrix, part# 00-0079 |
GeneArray Scanner 3000 | Affymetrix, part# 00-0212 |
Boiling water bath with floating rack | Various |
Hybridization consumables | |
Genflex Tag 16K Array v2 | Affymetrix, part# 511331 |
Denhardt’s Solution, 50X concentrate | Sigma, part# D2532 |
Streptavidin, R-phycoerythrin conjugate (SAPE) | Invitrogen, part# S866 |
Safe-Lock Microcentrifuge Tube, 0.5 mL | Eppendorf, part# 0030 123.301 |
Teeny Tough-Spots | Diversified Biotech, part# LTTM-1000 |
0.5 M EDTA | BioRad, part# 161-0729 |
10% Tween | Sigma, part# T2700 |
MES free acid monohydrate | Sigma, part# M5287 |
MES sodium salt | Sigma, part# M5057 |
5 M NaCl | Sigma, part# 71386 |
20X SSPE | Sigma, part# S2015 |
Molecular biology grade water | Sigma, part# W4502 |
Hybridization Primers | Various suppliers (standard desalting) |
Uptag | 5′ GATGTCCACGAGGTCTCT 3′ |
Buptagkanmx4 | 5′ biotin-GTCGACCTGCAGCGTACG 3′ |
Dntag | 5′ CGGTGTCGGTCTCGTAG 3′ |
Bdntagkanmx4 | 5′ biotin-GAAAACGAGCTCGAATTCATCG 3′ |
B213 | 5′ biotin-CTGAACGGTAGCATCTTGAC 3′ |
Uptagkanmx | 5′ GTCGACCTGCAGCGTACG 3′ |
Dntagkanmx | 5’GAAAACGAGCTCGAATTCATCG 3′ |
Uptagcomp | 5’AGAGACCTCGTGGACATC 3′ |
Dntagcomp | 5’CTACGAGACCGACACCG 3′ |
Upkancomp | 5’CGTACGCTGCAGGTCGAC 3′ |
Dnkancomp | 5’CGATGAATTCGAGCTCGTTTTC 3′ |
Sequencing Primers: Illumina Platform | Various suppliers |
UpTag Forward (100uM) | 5′ CAA GCA GAA GAC GGC ATA CGA GCT CTT CCG ATC T GAT GTC CAC GAG GTC TCT 3′ |
UpTag Reverse (100uM) | 5′ AAT GAT ACG GCG ACC ACC GAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T NNNNN GTC GAC CTG CAG CGT ACG 3′ |
DownTag Forward (100uM) | 5′ CAA GCA GAA GAC GGC ATA CGA GCT CTT CCG ATC T GAA AAC GAG CTC GAA TTC ATC G 3′ |
DownTag Reverse (100uM) | 5′ AAT GAT ACG GCG ACC ACC GAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T NNNNN CGG TGT CGG TCT CGT AG 3` |
Read 1 UP-tag seq primer (100uM) | 5′ GTC GAC CTG CAG CGT ACG 3′ |
Read 1 DOWN-tag seq primer (100uM) | 5′ CGG TGT CGG TCT CGT AG 3′ |
Read 2 Index Sequencing primer (standard Illumina R1 primer) (100uM) | 5′ AC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T 3′ |
Additional Sequencing Reagents/Equipment | |
Qiagen MinElute 96 UF PCR purification Kit | Qiagen, part# 29051 |
Vaccum pump | Any vendor |
Macherey-Nagel Vaccum Manifold | Macherey-Nagel, part# 740 681 |
Invitrogen Quant-iT dsDNA BR Assay Kit | Invitrogen, part# Q32853 |
Invitrogen Qubit assay tubes | Invitrogen, part# Q32856 |
40% acrylamide plus 1% N,N’-methylene-bis-acrylamide, 37.5:1 | Bio Rad, part# 161-0148 |
Tris Base | Sigma, part# T1503-1KG |
Boric Acid | Sigma, part# B6768-500G |
0.5M EDTA, pH 8.0 | Teknova, part# E0306 |
Ammonium Persulfate | Sigma, part# A3678-25G |
TEMED | Bioshop, part# TEM001.25 |
Ethidium Bromide Solution | Bioshop, part# ETB444.10 |
0.5M Ammonium acetate | Teknova, part# A2000 |
10mM Magnesium acetate tetrahydrate | Sigma, part# M0631-100G |
1mM EDTA, pH 8.0 | See 0.5M EDTA, pH 8.0 |
Ethanol | Various |
Sodium acetate, pH 5.2 | Teknova, part# S0297 |
Speed vacuum | Various |
Single-Read Cluster Generation Kit | Illumina, part# GD-300-1001 |
36c Sequencing Kit v4 | Illumina, part# FC-104-4002 |
10X TBE recipe
Amounts | Reagents |
108 grams | Tris Base |
55 grams | Boric Acid |
40mL | 0.5M EDTA (pH 8.0) |
Add dH2O to 1L mark |
12% Polyacrylamide gel recipe
Volumes | Reagents |
5.8 ml | 40% acrylamide plus 1% N,N’-methylene-bis-acrylamide, 37.5:1 |
12 ml | dH2O |
2 ml | 10X TBE |
140 μl | 10% Ammonium Persulfate |
Total volume: 20ml |
Uptag primer mix:
Resuspend Uptag and Buptagkanmx4 each in ddH2O at 100 μM, then mix in a 1:1 ratio for a final concentration of 50 μM each. Store at -20°C.
Downtag primer mix:
Resuspend Dntag and Bdntagkanmx4 each in ddH2O at 100 μM, then mix in a 1:1 ratio for a final concentration of 50 μM each. Store at -20°C.
Mixed oligonucleotides:
Resuspend each of the following eight oligos (standard desalted) in ddH2O at 100 μM:
Uptag, Dntag, Uptagkanmx, Dntagkanmx, Uptagcomp, Dntagcomp, Upkancomp, Dnkancomp.
Mix an equal volume of the eight oligonucleotides for a final concentration of 12.5 μM each.
12X MES stock:
For 10 ml, dissolve 0.7 g MES free acid monohydrate and 1.93 g MES sodium salt in 8 ml molecular biology grade water. After mixing well, check pH and adjust if needed to a pH 6.5-6.7. Add water to a total volume of 10 ml. Filter sterilize and store at 4°C protected from light (e.g., wrap the tube in foil). Replace if solution becomes visibly yellow or after 6 months.
2X Hybridization buffer:
For 50 ml, mix 8.3 ml of 12X MES stock (from 2.9.14), 17.7 ml of 5 M NaCl, 4.0 ml of 0.5 M EDTA, 0.1 ml of 10% Tween 20 (vol/vol), and 19.9 ml filtered ddH2O. Filter sterilize.
Wash A: Mix 300 ml 20X SSPE, 1 mL 10% Tween (vol/vol), 699 ml ddH2O. Filter sterilize.
Wash B: Mix 150 ml 20X SSPE, 1 mL 10% Tween (vol/vol), 849 ml ddH2O. Filter sterilize.
Barcode sequencing primers
In UP-tag primer sequences the 5′ tail (bold) are Illumina specific adaptor sequences incorporated into the F and R primer. The variable sequence (italics) represents the 5-mer indexing tag used in multiplexing/ index read-out. The 3′ tail (underlined) represents the common primer flanking the uptag barcode and is required to amplify the yeast barcodes.
In DOWN-tag primer sequences the 5′ tail is identical to 5′ tail of UP-tag primers (Illumina specific sequence), however, the 3′ tail (underlined) is replaced with the common primers that are used to amplify the DOWN-tag barcodes.