nextflex® chip-博天堂备用

nextflex™ chip-seq kit是为起始量低的样本进行建库而设计的一款文库制备试剂盒，样本起始量低至1ng的chip dna或genomic dna，兼容于illumina 测序平台。

nextflex chip-seq kit使用bioo专利enhanced adapter ligation technology，并以较少的样本起始量得到较高的文库产量。连接酶预混液的功效进一步提升，用户可有效的连接更长的接头，获得更好的连接效果。nextflex chip-seq kit采用酶预混试剂和磁珠纯化法，简化了整个实验流程，减少移液步骤和建库消耗的时间。

nextflex™ chip-seq barcodes与enhanced adapter ligation technology搭配使用，用户可同时处理96种样本来提供chip测序能力。

 

产品特点：

• 样本dna起始量低至1-10ng 
• 高效率的nanoq™ 连接酶，提高了接头连接率，降低建库样本起始量
• 适用于chip, genomic dna 或cdna样本
• 整个实验过程更加快速—磁珠法纯化节省了建库时间
• 实验方案消除对凝胶纯化的需求
• 匹配多种自动化建库系统
• 灵活的接头方案供选择—试剂盒包含6、12、24、48和96重独特barcodes
• 经illumina测序平台验证

 

产品列表：

货号	产品名称	规格
nova-5143-01	nextflex® chip-seq library prep kit  for illumina® sequencing	8 rxns
nova-5143-02	nextflex® chip-seq library prep kit  for illumina® sequencing	48 rxns
nova-514120	compatible chip-seq barcodes 6 barcodes	48 rxns
nova-514121	compatible chip-seq barcodes 12 barcodes	96 rxns
nova-514122	compatible chip-seq barcodes 24 barcodes	192 rxns
nova-514123	compatible chip-seq barcodes 48 barcodes	384 rxns

 

nextflex chip-seq kit protocol：

 

achieving high coverage & yield from gc & at rich genomes

standard next generation sequencing (ngs) library preparation methods include a polymerase chain reaction (pcr) amplification step prior to cluster generation and sequencing, to meet minimum molarity cutoffs required by different sequencing platforms. biases associated with pcr amplification include uneven coverage of regions with extreme base composition, increased numbers of duplicate fragments, decreased mapping quality and poor variant calling. particularly intractable regions of extreme base composition include gc-rich regions, which remain difficult to uniformly pcr amplify. while the human genome is not considered gc-rich (3.2 gb; 41% gc), certain regions of the genome can be extremely gc rich, e.g. retinoblastoma tumor suppressor gene rb1 (104 of the first 136 coding bases are either g or c). unbiased amplification of highly complex whole genome sequencing (wgs) libraries, whole exome sequencing (wes) libraries and targeted re-sequencing libraries is required to obtain high quality data for de novo genome assembly and accurate calling of single nucleotide polymorphisms (snps). here, we show that the nextflex™ pcr polymerase is a leader in providing high quality amplification. the nextflex pcr polymerase, used in all of the nextflex dna-seq and rna-seq ngs library preparation kits, contains a highly optimized and robust enzyme that exhibits minimal gc bias and produces uniform coverage of difficult to sequence genomes.

methods

genomic dna was isolated from deinococcus radiodurans (3.2 mb; 67% gc) and saccharomyces cerevisiae (12.1 mb; 38% gc). genomic dna was covaris sheared to an average fragment size of 200 bp. 20 ng of d. radiodurans (gc-rich) and s. cerevisiae (at-rich) dna was used as starting material for library preparation using the nextflex™ chip-seq kit, following the gel-free protocol. following barcoded-adapter ligation, gc-rich and at-rich dna samples were divided equally and independently pcr amplified for 14 cycles according to manufacturer’s instructions. each library was prepared in duplicate. average library size was assessed by bioanalyzer® and concentrations determined by qpcr. normalized libraries were clustered on-board, and 150 bp paired-end sequencing was performed on the hiseq® 2500 across 4 lanes of 2 flowcells using rapid run mode.

11 different pcr enzymes used in library preparation kits for next generation sequencing were tested in this sequencing experiment. the performance of the three best are highlighted here: nextflex pcr polymerase, supplier d and supplier i. this sub s  et of enzymes produced the most consistent metrics, including library yield, gc bias and raw coverage, of the 11 pcr kits tested across two genomes. quality control sequencing metrics against the at-rich genome show more paired-end reads for the nextflex pcr polymerase, and more total alignments and non-duplicated fragments than suppliers d and i (table 1).

results

library yield and complexity

several metrics, including library yield, were utilized to compare polymerase performance. achieving sufficient library yields is important in order to meet minimum molarity cutoffs required by different sequencing platforms. each polymerase produces distinct library quantities (figure 1). library yield comparison across the at-rich genome shows a similar trend: libraries pcr amplified using nextflex pcr polymerase or supplier d’s enzyme produce almost two times more library product than supplier i. conversely, across the gc-rich genome, nextflex pcr polymerase and supplier i are equal; however, supplier d library yields are reduced. this result shows the robust ability of the nextflex pcr polymerase to equally amplify both gc-rich and at-rich genomes, and the inconsistent pcr efficiency of suppliers d and i when using the same template for pcr. library yields are important, but can give rise to misleading interpretations. library yield inconsistencies produced by supplier d suggest an unequal amount of at-rich over gc-rich clones. although library yields for supplier d over the at-rich genome are roughly equal to library yields of nextflex pcr polymerase (figure 1), sequence complexity and richness (table 1) are lower, while gc bias is higher and coverage is uneven (figures 2, 3 and 4). the same trend is evident for supplier i (figure 2, 3 and 4).

gc bias 

to obtain a global representation of each pcr enzyme’s effect on sequence integrity, we examined pcr-introduced gc bias across both s. cerevisiae and d. radiodurans genomes using picard gc bias plots (figure 2). low gc bias would produce a normalized coverage slope of one, or a horizontal line over windows of gc-rich content. alternatively, high gc bias would produce a normalized coverage slope greater than one, over windows of gc-rich content (figure 2, s. cerevisiae, supplier i). picard analysis shows nextflex pcr polymerase to be the most consistently unbiased polymerase tested, regardless of genomic complexity and extreme base composition. across the s. cerevisiae genome, nextflex pcr polymerase showed the lowest level of gc bias, with close to mean coverage across the entire genome, including gc-rich and at-rich stretches. unlike the nextflex pcr polymerase, normalized coverage of supplier i across the s. cerevisiae genome is noticeably biased and uneven, producing uneven coverage of the genome, with fewer reads mapping to gc-rich regions and higher levels of pcr duplication in at-rich regions. conversely, the most average normalized coverage across the d. radiodurans genome was produced by supplier i. nextflex pcr polymerase and supplier d had slightly higher gc bias over gc-rich windows of the genome. overall, however the nextflex pcr polymerase provides a balanced representation of both at-rich and gc-rich regions, which neither polymerase d or i attains.

genome coverage

in addition to gc bias, coverage across average gc-rich and at-rich regions was considered for each polymerase. across all four chromosomes of the d. radiodurans genome, nextflex pcr polymerase consistently outperforms suppliers d and i, with higher levels of unbiased coverage (figure 3). the nextflex pcr polymerase produced 110-160x coverage of the entire d. radiodurans genome compared to 75-110x coverage and 95-130x coverage for suppliers d and i, respectively. similar to coverage data of the gc-rich genome, the nextflex pcr polymerase shows higher coverage of the first eight chromosomes of the at-rich genome than both suppliers d and i (figure 4; coverage of chromosomes 1-8 shown, 9-16 showed similar results). the nextflex pcr polymerase produced 30-40x coverage of the first eight chromosomes of the s. cerevisiae genome, compared to 25-35x coverage and 20-25x coverage for suppliers d and i, respectively.

conclusions

our comparison between the nextflex pcr polymerase and those of suppliers d and i found the nextflex pcr polymerase, which is used in all of the nextflex dna library prep kits, including the nextflex chip-seq kit, and rna-seq ngs library preparation kits, to be the most unbiased enzyme for ngs library prep across two highly diverse genomes, while consistently delivering even and high read coverage. consistency of library yield between these two genomes demonstrates that this enzyme is extremely robust and can be applied to numerous ngs applications including, but not limited to, wgs, wes, metagenomics, target sequencing and other uses.

 

kit contents

• nextflex® chip end repair buffer mix
• nextflex® chip end repair enzyme mix
• nextflex® chip adenylation mix
• nextflex® chip ligation mix
• nextflex® chip adapter (0.6 µm)
• nextflex® chip primer mix (12.5 µm)
• nextflex® chip pcr master mix
• nuclease-free water
• resuspension buffer
• chip-seq dna control

required  materials not provided

• 1 ng – 10 ng of dna in up to 40 µl nuclease-free water
• nextflex® chip-seq barcodes – 6 / 12 / 24 / 48
• ethanol 100% (room temperature)
• ethanol 80% (room temperature)
• covaris system (s2, e210) or other device for fragmenting dna
• 96 well pcr plate non-skirted (phenix research®, cat # mps-499) / or / similar
• 96 well library storage and pooling plate (fisher scientific®, cat # ab-0765) / or / similar
• adhesive pcr plate seal (bio-rad®, cat # msb1001)
• agencourt® ampure® xp 5 ml (beckman coulter genomics®, cat # a63880)
• magnetic stand -96 (thermo fisher® scientific, cat # am10027) / or / similar
• heat block
• thermocycler
• 2, 10, 20, 200 and 1000 µl pipettes / multichannel pipettes
• nuclease-free barrier pipette tips
• microcentrifuge
• 1.5 ml nuclease-free microcentrifuge tubes
• vortex

nextflex chip-seq kit 和nextflex™ chip-seq barcodes部分引用文献：

• fischer, n., et al. (2014) rapid metagenomic diagnostics for suspected outbreak of severe pneumonia [letter]. emerg infect dis. doi: 10.3201/eid2006.131526.
• gosselin, d. et al. (2014) environment drives selection and function of enhancers controlling tissue-specific macrophage identities. cell. doi:10.1016/j.cell.201411023.
• golomb, b. l., hirao, l. a., dandekar, s. and marco, m. l. (2016) gene expression of lactobacillus plantarum and the commensal microbiota in the ileum of healthy and early siv-infected rhesus macaques. scientific reports. doi:10.1038/srep24723.
• lelandais, g., blugeon, c. and merhej, j. (2016) chipseq in yeast species: from chromatin immunoprecipitation to high-throughput sequencing and bioinformatics data analyses. yeast functional genomics: methods and protocols, methods in molecular biology, 1361. pg 1-11. springer science business media new york. doi: 10.1007/978-1-4939-3079.