LabLog-SFL

LogID	Title	Initial	Date
AF001	Codebook for GA341 (= YJC AE538 = YMU RNA-seq on OKB2/S1F/THP1)	SFL	10/21/15
AF002	Experimental sheme for AE592 (YMU RNA-seq GA416 and GA417)	SFL	10/23/15
AF003	Codebook for GA417	SFL	11/3/15
AF004	Codebook for GA416	SFL	11/4/15
AF005	Codebook for Po-Ting	SFL	11/5/15
AF006	Codebook for tidy 40T/N raw data (2015_1110_11SVA_40TN_raw = 40TNRaw)	SFL	11/10/15
AF007		SFL

The history of primary infection of EBV in keratinocytes

2006/10

Molecular and cytogenetic changes involved in the immortalization of nasopharyngeal epithelial cells by telomerase (the link to NCBI)

2008/10/10

根據這一篇, 那送EBV進入NP460h-hTERT不就等同於把EBV關禁閉? 另外Dr.George Miller有篇在J.Virol.Methods(2006)的文章,看起來像是一位MD/PhD的學生做的。他以兩位IM病人血清(富含各種anti-EBV抗體)去分離經NaB處理過的P3HR1(HH514)細胞。分什麼呢?分那些在走lytic cycle和不被NaB活化的HH514。分下來後的細胞再送回去petri-dish養。結果發現走lytic cycle的細胞無一存活、而沒有induce的細胞可以再被養回來,而且induction rate隨著時間增加而增加。很有意思的一篇文章。George大學是學歷史的,非常會寫文章,邏輯性很強而且要言不贅,讀他的paper總覺得能做science真好。

LabLog-柏廷篇

LogID	Title	Initial	Date
AE801	Compare Serum folate concn in OPMD patient and summary MAF in SNPs of folate-mediated one carbon metabolism	PTL	2015/11/9
AE802	Calculate and compare the MAF of rs1801133 and rs1801131 from sample	PTL	2015/11/13
AE803	Calculate and compare the MAF of rs2274976, rs1805087, rs1801394 and rs1979277 from sample	PTL	2015/11/17
AE804		PTL
AE805		PTL

#1由 poting 在 三, 11/11/2015 - 09:14 發表。

修改完畢

老師 我已經修改完畢了

#2由 poting 在 一, 11/09/2015 - 12:06 發表。

AE801 result

•FTEST=0.039 < 0.05      Variance between control and OPMD

•P=0.067 > 0.05               No significant difference  

IRB 問與答 2014年題庫

IRB 問與答 2014年題庫 由 sufang 在 二, 06/09/2015 - 09:09 發表  Oral Cancer   IRB   Oral Cancer

Su-Fang Lin <sflin1@gmail.com> Jun 5 Reply to 張書銘, 陳雅雯, 江士昇, 夏興國 
Dear All,

我PP計畫的IRB審查回來了, 兩個reviewer都有一點問題. 總評皆為「修正後推薦」。想請問大家先前有沒有碰到類似的問題, 其正解為何? 先謝謝大家的幫忙!  之後如果大家不介意的話, 我們是不是把各自的問題集合在一起、弄成一本題庫? 具IRB同仁說 送出去的審委不一樣、同樣的申請件, 碰到的問題都不一樣。  Thanks again!  Su-Fang (ext 35107)   (餘刪, 見下6/8回覆)

---

Ya-Wen Chen Jun 5 Reply to me, 張書銘, 江士昇, 夏興國 

以下是我遇到的問題及回答, 素芳的問題與我的問題有相似, 重點是(簡易審查核對表)提及使用合法生物資料庫..
簡易審查核對表我更改成接續前階段研究之後續資料分析(湘如建議).
 

EBV and NGS

由 sufang 在 四, 06/04/2015 - 13:38 發表  Pre-published   EVKV   NGS

3552_2012_12_Whole genome sequencing of the Akata and Mutu Epstein-Barr virus (EBV) strains.pdf

3577_2011_11_Direct sequencing and characterization of a clinical isolate of Epstein-Barr virus from nasopharyngeal carcinoma tissue by using next-generation sequencing technology

3964_2014_02_Integrated mRNA and microRNA transcriptome sequencing characterizes sequence variants and mRNA–microRNA regulatory network in nasopharyngeal carcinoma model systems

2015/05 Methylation Marker Paper from JAMA Oncology

Correlation of Smoking-Associated DNA Methylation Changes in Buccal Cells With DNA Methylation Changes in Epithelial Cancer JAMA Oncol. Published online May 14, 2015. doi:10.1001/jamaoncol.2015.1053

Andrew E. Teschendorff, PhD^1,2; Zhen Yang, PhD²; Andrew Wong, PhD^3; Christodoulos P. Pipinikas, PhD⁴; Yinming Jiao, MSc²; Allison Jones, BSc⁵; Shahzia Anjum, PhD⁵; Rebecca Hardy, PhD³; Helga B. Salvesen, MD⁶; Christina Thirlwell, PhD⁴; Samuel M. Janes, PhD⁷; Diana Kuh, PhD³; Martin Widschwendter, MD⁵

---

At a Glance

¶ The purpose of the research was to assess the suitability of buccal cells as an epithelial source of tissue to examine the effects of smoking on the epigenome, and to test whether these effects are also seen in smoke-related epithelial cancers.

¶ Smoking is associated with widespread changes in the DNA methylation landscape of buccal cells.

¶ Some smoking-associated DNA methylation changes are common to buccal and blood tissue, but buccal cells exhibit significantly more changes than blood cells.

¶ Smoking-associated DNA methylation changes in buccal cells correlate with DNA methylation changes in epithelial cancers and do so most strongly in smoke-related epithelial cancer.

2016/105年待辦事項

2016/105年待辦事項

由 sufang 在 三, 05/27/2015 - 08:45 發表 Pre-published Gene Fusion HOK Lin Lab Oral Cancer

Paper Submission

宛樺: DDR1 overexpression in OSCC 

嬿如: EBV Rta mediated DNA methylation in the host genome

 

FGFR Gene Fusion related (玉蓮、奕宏)

1. FGFR2 gene fusion in cholangiocarcinoma (Post 連結)

2. FGFR3 gene fusion in oral cancer (Post 連結)

 

Carcinogen induced epigenetic transformation in human oral keratinocytes (嬿如、英潔)

1. Molas analysis of RNA-Seq data (Post 連結)

2. MTHFR SNPs and epigenetic transformation

3. Develop methylation markers for head and neck patients

 

Mutational landscape of cholangiocarcinoma (Post 連結)

 

Mutational landscape of OSPCC and Oral Cancer (Post 連結)

 

Grant Proposal and Report

1. 104-CAPP05

    月報 x 12 (in progress)

    期中報告 (done by 佩瑩)

    期末報告

2. 105-CAPP  執行與考核點設定

3. 106-CAPP  計畫書撰寫

4. MOHW 衛福部OPMD計畫

    實驗進行 (記得送OSCC DNA給Jeffrey Lab. 一併測試MTHFR)

5. 105-MOST 科技部計畫 (11月中 call for proposal)

6. 105-MOST PPG (LOI submitted)

Reading Notes

Wiki  Biological action (2015/05/17)

In many Asian cultures, the areca nut is chewed along with betel leaf to obtain a stimulating effect. Arecoline is the primary active ingredient responsible for the central nervous system effects of the areca nut. Arecoline has been compared to nicotine; however, nicotine acts primarily on the nicotinic acetylcholine receptor. Arecoline is known to be a partial agonist of muscarinic acetylcholine M1, M2, M3 receptors and M4 which is believed to be the primary cause of its parasympathetic effects (such as pupillary constriction, bronchial constriction, etc.).

LD50: 100 mg/kg, administered subcutaneously in mouse.

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2015/05/25

pdf 4377 2014-Feb New nucleophilic mechanisms of ROS-dependent epigenetic modifications: comparison of aging and cancer

Igor Afanas'ev Vitamine Research Institute, Moscow, Russia, Poetom Portugal

ROS-induced DNA Methylation in Cancer 共舉12例

2015-05-05 Journal Club

各位老師學長學姊大家好:

我是下周二要在journal club報告的學生-Yi-Hong

題目:

Massive parallel sequencing uncovers actionable ​FGFR2–​PPHLN1 fusion and ​ARAF mutations in intrahepatic cholangiocarcinoma

本文文章

supplementary information

RBBS

RBBS
由 sufang 在 三, 04/01/2015 - 09:00 發表 Pre-published RBBS

須細讀, 台大擬用此法screen HNSCC patients.

Start: 04/01/2015

2015-04-21 Journal Club Paper

大家好: 這篇是我下周要報的paper

感謝!

OSCC Cell Line STR information 20150312 更新版

OSCC Cell Line STR information 20150312 更新版
由 sufang 在 一, 03/30/2015 - 08:45 發表 Oral Cancer OSCC Cell Line STR analysis

Dear all,

更新oral cancer相關細胞株之STR資訊
共有16個細胞株, 其中DOK, FaDu, HOK, HSC-3, KB, OEC-M1, SCC-15, SCC-9, Tu-183(共9株)有送件STR的資訊(詳如附件)
須請大家注意的資訊如下:
1. HSC-3, 於Dr.郭靜娟's lab的新HSC-3為真, 請非此來源者把舊的細胞扔了吧! (p.s Dr. 李岳倫於2014年7月及9月分別定的STR結果同新HSC-3, 推測亦為可用細胞株)
2. KB, 於Dr.郭靜娟's lab定STR結果為HeLa細胞, ATCC/JCRB資料庫也證明KB為HeLa. 請勿使用此細胞株!
3. SCC-15, Dr.李岳倫's lab 於2014年9月及10月分別定的STR結果皆與SAS細胞株相符(90.9% match), 使用此細胞株請小心!
4. Tu-183, 於Dr.江士昇's lab於2014年12月所定STR結果未比對到現今資料庫中任何細胞株, 推測可能為可用細胞株. 但Dr. 江提供資訊:expression pattern與預期不同, 大家使用此細胞株請小心!

以上,
如有問題或錯誤的地方, 敬請不吝指正! 謝謝!

敬祝 研究順利

嬿如 敬上

原檔按我下載 (2015_0320_cell_lines_str_information20150312ding_gao.xlsx)

大家講過的Paper大全

2015/03/24 陳貞佩

Monensin inhibits epidermal growth factor receptor trafficking and activation: synergistic cytotoxicity in combination with EGFR inhibitors

2015/03/10 楊韻涵 

NRF2-driven miR-125B1 and miR-29B1 transcriptional regulation controls a novel anti-apoptotic miRNA regulatory network for AML survival

2015/02/10 王思涵

Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration 

2015/01/06 小嬿

RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

2015-03-24 Journal Club Paper

Dear all:

附件為我下週要報告的paper
請查收,謝謝!

貞佩

Monensin inhibits epidermal growth factor receptor trafficking and activation: synergistic cytotoxicity in combination with EGFR inhibitors

2015-03-10 Journal Club Paper

各位老師、學長學姊大家好:
附檔是我這周要報告的paper
請查收 謝謝大家

祝 研安
韻涵 敬上

NRF2-driven miR-125B1 and miR-29B1 transcriptional regulation controls a novel anti-apoptotic miRNA regulatory network for AML survival

2015/01/30 國長會議

由 sufang 在 五, 03/13/2015 - 08:29 發表 Pre-published 國長會議 長庚

國衛院長庚雙邊會議 2015/01/30 (五) at 林口長庚

王陸海: miR491-3p is responsible for oral cancer metastasis
葉大森: bile duct cancer (miRNA 200下降), liver cancer (miRNA 200 上升，which is correlated with EMT)
陳鈴津教授介紹 閻紫宸教授另外一個專長是 radio pharmatheutical research (新藥開發 GMP等等)
-----------------------
趙盈凱 副教授【胸腔外科】可吸收性生物醫材在食道癌之應用
東方人食道癌 是由ABC引起的、西方的食道癌則多由胃食道逆流
證實CCRT+Surgert is better therapy than just surgery
-Big Database (2400 病人 由該醫師和助理慢慢build up)
-健保加值資料庫
-cisplatin membrane
biodegradable polymers in thoracic disease
-----------------
陳立宗 特聘研究員兼所長 【國衛院癌症研究所】 Pancreatobiliary Cancer Clinical Trial in Taiwan
–TCOG很多東西需要長庚醫院幫忙 e.g. 陳敏夫教授 adjuvant interferon alpha 2b TCPG T1297 (this is a negative study, indicating that Taiwanese surgery was very successful)
–原來文獻建議 肝癌患者 開完刀後若能 加上抗病毒藥物治療 可有效降低recurrence rate.
但是由TCOG的結果看來, not necessary.
-----------------

ds-cDNA prep for gene fusion detection

3/6/2015 DR 提醒

1: Daw-Yang seems to use Clontech ds-cDNA prep for single cell Seq + Tagmentation

2: There is no way to add specific primer in this way...

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According to Daw-Yang:  I will use Nexera XT for DNA prep.

Figure 1 AMP for targeted RNA and DNA sequencing (pdf 4231). 

Double-stranded cDNA (dscDNA) synthesis starts with total nucleic acid or RNA from fresh or FFPE material without ribosomal RNA or genomic DNA(gDNA) depletion. Solid-phase reversible immobilization (SPRI)-cleaned double-stranded cDNA or fragmented genomic DNA is processed with end repair and dA tailing, directly followed by ligation with a half-functional adapter. SPRI-cleaned, ligated fragments are amplified with 10–14 cycles of multiplex PCR1 using gene-specific primers (GSP1 pool) containing a PCR multiplexing tag (brown) and a primer complementary to a portion of the universal ligated adapter (short green). An unknown fusion partner or target sequence is indicated by a question mark. SPRI-cleaned PCR1 amplicons are amplified with a second round of 10-cycle multiplex PCR2 using a combination of GSP2 pool nested gene-specific primers (3' downstream of GSP1), which are tagged with the second adapter sequence specific for Ion Torrent (black-blue) or Illumina (black-red, subsequently tagged with an indexing primer (red-orange-purple)), and a second nested primer against the ligated universal adapter (long green). After a final SPRI cleanup, the target amplicon library is ready for quantitation, downstream clonal amplification and sequencing. 

 

 

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Fluidigm 4-Primer Amplicon Tagging

The Access Array 4-primer amplicon tagging scheme is based on a step-out four primer PCR strategy where tagged target-specific (TS) primer pairs are combined with sample-specific primer pairs that contain a barcoding sequence and the adaptor sequences used by Illumina sequencing systems. By incorporating sample-specific barcodes, all 2,304 PCR products generated from a 48.48 Access Array Integrated Fluidic Circuit (IFC) are unique and can be pooled together to run in a single sequencing experiment.

 

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Nextera_xt_sample_prep

TCGA HNSCC Marker Paper

由 sufang 在 五, 02/27/2015 - 10:08 發表 Oral Cancer HNSCC TCGA

Comprehensive genomic characterization of head and neck squamous cell carcinomas (pdf 4292; PubMed Link)

The Cancer Genome Atlas profiled 279 head and neck squamous cell carcinomas (HNSCCs) to provide a comprehensive landscape of somatic genomic alterations. Here we show that human-papillomavirus-associated tumours are dominated by helical domain mutations of the oncogene PIK3CA, novel alterations involving loss of TRAF3, and amplification of the cell cycle gene E2F1. Smoking-related HNSCCs demonstrate near universal loss-of-function TP53 mutations and CDKN2A inactivation with frequent copy number alterations including amplification of 3q26/28 and 11q13/22. A subgroup of oral cavity tumours with favourable clinical outcomes displayed infrequent copy number alterations in conjunction with activating mutations of HRAS or PIK3CA, coupled with inactivating mutations of CASP8, NOTCH1 and TP53. Other distinct subgroups contained loss-of-function alterations of the chromatin modifier NSD1, WNT pathway genes AJUBA and FAT1, and activation of oxidative stress factor NFE2L2, mainly in laryngeal tumours. Therapeutic candidate alterations were identified in most HNSCCs.

Five Figures in the Main Text:

Figure 1 | DNA copy number alterations. a, Copy number alterations by anatomic site and HPV status for squamous cancers. Lung squamous cell carcinoma (LUSC, n = 358) and cervical squamous cell carcinoma (CESC, n = 114). b, Unsupervised analysis of copy number alteration of HNSCC (n = 279) with associated characteristics. The rectangle indicates chromosome 7 amplifications in the purple cluster. NA, not available. 

Figure 2 | Significantly mutated genes in HNSCC. Genes (rows) with significantly mutated genes (identified using the MutSigCV algorithm; q , 0.1) ordered by q value; additional genes with trends towards significance are also shown. Samples (columns, n = 279) are arranged to emphasize mutual exclusivity among mutations. Left, mutation percentage in TCGA. Right, mutation percentage in COSMIC (‘upper aerodigestive tract’ tissue). Top, overall number of mutations per megabase. Colour coding indicates mutation type.

Figure 3 | Candidate therapeutic targets and driver oncogenic events. Alteration events for key genes are displayed by sample (n = 279). TSG, tumour suppressor gene.

Figure 4 | Integrated analysis of genomic alterations. a,b,Samples (n=279) ared isplayed in columns and grouped by gene expression (a) or methylation (b) subtype (sub.). Unadjusted two-sided Fisher’s exact test P values assess the association of each genomic alteration. Methylation probe location of CpG islands, shores and shelves are shown on the left of b. Annotation shows HPV status and subtype (16, 33 and 35). CN, copy number.

Figure 5 | Deregulation of signalling pathways and transcription factors. Key affected pathways, components and inferred functions, are summarized in the main text and Supplementary Information section 7 for n = 279 samples. The frequency (%) of genetic alterations for HPV(–) and HPV(+) tumours are shown separately within sub-panels and highlighted. Also see Supplementary Fig. 7.15. Pathway alterations include homozygous deletions, focal amplifications and somatic mutations. Activated and inactivated pathways/genes, and activating or inhibitory symbols are based on predicted effects of genome alterations and/or pathway functions. 

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Ten Parts of Supplementary Information: 

S1: Biospecimen collection and clinical data

S1.1. Biospecimen collection and clinical data  

S1.2. HPV detection methods  

S1.3. Survival analysis  **

Figure S1.1. HPV status as a function of clinical and molecular characteristics  

Figure  S1.2.  Receiver  operating  characteristic  (ROC)  curves  in  HPV-associated  miRNAs  in  oropharyngeal HNSCC  

Figure S1.3. DNA methylation signatures of HPV

Figure S1.4. Survival analysis for select clinical and genomic variables

Figure S1.5. Survival analysis for platform-specific subtypes 

Table S1.1. Summary of clinical data 

Data file S1.1. Data freeze clinical dataset (This file contains clinical and demographic information for all patients)

Data file S1.2. Summary of HPV detection results  (This file contains the results of molecular analyses used to determine the HPV status of all patients. These include data from in situ hybridization, p16 staining, RNA and DNA sequencing, and the MassArray assay.)

Data file S1.3 Mutation signatures by HPV status (This file contains counts of base changes seen in APOBEC and smoking mutation signatures for HPV(+) and HPV(-) subjects. Fisher’s exact test p-values are also shown.)

S2: Copy number analysis  

S2.1. SSNP array-based copy number analysis

S2.2. Structural alterations  

Figure S2.1. GISTIC 2.0  analysis  of  significantly  reoccurring  focal  alteration  in  279  HNSCC tumors  

Figure S2.2. GISTIC amplification and deletion peaks in lung squamous cell and cervical squamous cell  carcinoma  

Figure S2.3. Comparison of GISTIC 2.0 analyses of 243 HPV(–) and 36 HPV(+) head and neck tumors  

Figure S2.4. Number of copy number segments in HPV(+) and HPV(–) samples  

Data file S2.1. GISTIC amplification and deletion peak annotation in head and neck squamous cell (all  samples, by HPV

status, and by site), lung squamous cell carcinoma, cervical squamous cell carcinoma  (results from GISTIC analyses for head and neck (HNSC), lung, and cervical squamous cell carcinoma. Additional analyses for HNSC were conducted by HPV status and tumor site.)

Data  file  S2.2. Fisher’s  exact  test  p-values  for  frequency  comparisons  of  significantly  reoccurring  alterations by HPV

status and site (results of analyses of frequency comparisons of significantly reoccurring DNA copy number alterations by HPV status and tumor site. Fisher’s exact test p-values are also shown.)

S3: RNA sequencing  

S3.1. RNA sequencing and expression quantification  

S3.2. RNA-Seq for confirmation of somatic alterations reported in whole exome sequencing  

S3.3. Gene fusion detection  

S3.4. RNA-Seq for gene splicing and viral integration  

Figure S3.1. RNA-Seq for confirmation of somatic alterations reported in whole exome sequencing  

Figure S3.2.  FGFR3-TACC3 fusion event  

Figure S3.3. EGFR vIII mutant sample  

Figure S3.4. Exon 14 skipping in MET+ 

Figure S3.5. Alterations of CDKN2A gene structure, copy number, and expression of its protein coding  transcripts p16INK4A and p14ARF  

Figure S3.6. Integration of DNA mutation type, copy number, and gene expression for CDKN2A+ 

Figure S3.7. Alterations of FAT1 gene structure, copy number, and expression  

Figure S3.8. Integration of DNA mutation type, copy number, and gene expression for FAT1+ 

Figure S3.9. Integration of DNA mutation type, copy number, and gene expression for predicted driver  genes relevant to HNSCC  

Figure S3.10. Distribution of HPV integration breakpoints across the host genome

Figure S3.11. The KLK12 gene documents recurrent alternate transcription in HNSCC  Figure S3.12. Heterogeneous TP63 isoform usage in HNSCC+ 

Data file S3.1. RNA-Seq predicted fusions ( contains the results of fusion detection analyses performed with MapSplice. )

Data file S3.2. Viral integration sites (information about HPV viral integration sites based on the analysis of RNA and DNA sequencing 
data.)

Data file S3.3. SigFuge clustering results for alternatively spliced genes (the results of SigFuge analyses to detect differential expression of multiple gene isoforms. Uncorrected p-values are shown.)

Molas Analysis on Arecoline-Treated HOK Cells

02/16/2015

MTHFR, SHMT1, SHMT2

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02/17/2015

1. Library comparison

2. OKB2/AC (>=0.1) combined with OKB2/untreated (>=1), >= 2-fold (2886 transcriptid), sent to GO

Biological Process (BP): GOBP

Molecular Function (MF): GOMF

Cellular Compartment (CC): GOCC

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03/28/2015

Excel 製圖時

1. On "OKB2"  Filter > 3, shade them

2. On "Fold OKB2" Filter >=1.5 shade with red; Filter <=0.67 shade with blue

3. On "S1F"  Filter > 3, shade them

4. On "Fold S1F" Filter >=1.5 shade with red; Filter <=0.67 shade with blue

5. On "THP1"  Filter > 3, shade them

6. On "Fold THP1" Filter >=1.5 shade with red; Filter <=0.67 shade with blue

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04/02/2015 

感謝大家的製圖與整理!   再來要做啥?  我有幾個想法

1. Nucleosome assembly的原料, i.e. histones, 全面下降. 表示細胞若正在S phase, 再來的nucleosome assembly將有巨大變化. 是符合hypomethylation嗎?

2. 我們map出來的histone gene多在ch6, 少數幾個在ch1. 這些cluster在一起的gene family, 使要被調降的?

3. 還有沒有其它類似例子?

4. 如果ROS是元凶 (許多AC造成的上升或下降, 可被GSH reverse 回來), 機制是什麼? 

5. To confirm or extend more experiments, let's try on OKF4-hTert, NP460hTert and CGHNK2.

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04/14/2015 

1. Focus on nucleosome downregulation: histone cluster 1 on ch6 and histone cluster 2 on ch1.

2. Large hypo-methylation blocks: 在正常細胞時多為LAD/LOCK domain, 經過"transformation stimuli"後, 成為hypo-methyl region.

    LAD: lamina-associated domain

    LOCK: laroge organized chromatin lysine-modification

3. 40T/N methylation array中, delta beta >= 0.35 定義為 hypermethylated genes, delta beta <= -0.3 定義為 hypomethylated genes.

4. 40T/N methylation array中: 

    N_MAX-N_MIN  delta beta >= 0.3 計有6830 (表示 normal tissue中, methylation差異性的存在)

    T_MAX-T_MIN   delta beta >= 0.3 計有10808 (表示 tumor tissue中, methylation差異性的存在)
    T AVB-N AVB  delta beta >= 0.3 計有702 (Illumina廠商根據其它條件 例如是否有CpG island, 再縮減成37個up, 37個down.

5/26/2015 ^New

On Molas portal:

step 1: OKB2 > 1, OKB2_AC > 0,   Fold >= 2, 得 3220 transcripts.

step 2: OKB2 > 0, OKB2_AC >= 2, Fold >= 2 , 得1460 transcripts.

step 3: combine step 1 and step 2, 用 "IF" 公式 , 得 unique transcrpt n = 3563.

step 4: Sort for value, < 0.1者全部replace為0.1, 計算Fold.

step 5: Sort for GeneName / OKB2值大到小 / OKB2_AC 值大到小, 用"IF" 公式過濾unique gene name.

小計: unique gene name n= 2547 (down 1873, up 674).

         unique transcript / no gene name n=155.  

         2547 + 155 = 2702 = 1987 down, 715 up.

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Among the 2547 unique genes, we will focus on

(1) chromatin organization genes

(2) cell cycle related genes

(3) TP53 related genes (TP53值較高的transcript 因未達2倍差異, 反未被挑出 )

(2) anti-oxidant genes

(3) secreted proteins 

寄送SW780 RNA給長庚及行動基因陳華鍵

寄送SW780 RNA給長庚及行動基因陳華鍵
由 sufang 在 五, 02/13/2015 - 15:20 發表 Cholangiocarcinoma GeneFusion

From: Su­Fang Lin [mailto:sflin1@gmail.com] Sent: Friday, January 30, 2015 2:06 PM
To: angel945@cgmh.org.tw
Subject: this is Su­Fang's email

Dear Dr. 趙:
This is my email. I will prepare ~5­10 micrograms RNAs from SW780 cells for

your control. Please let me know to whom and to where should I sent. Thanks!

Best, Su­Fang 

--------------------------------------

Angel Chao Fri, Jan 30, 2015 at 2:49 PM To: Su­Fang Lin
Cc: 蔡佳龍 , knoxtn@cgmh.org.tw

Dear Prof. Su‐Fang,

Thank you very much indeed for your prompt e‐mail. Dr. Tsai (蔡佳龍博士)will give you the details for RNA transfer to our lab. Thanks again and nice meeting you at this fruitful CGMH‐NHRI meeting.

Regards, Angel

-----------------------------------------

蔡佳龍 Fri, Jan 30, 2015 at 4:16 PM To: Angel Chao
Cc: Su­Fang Lin , 王子豪醫師

林老師您好: 非常感謝您願意提供給我們SW780 cell RNA

由於RNA運送須以乾冰低溫運送比較麻煩 不知能否冒昧的跟老師要SW780 cell, 以常溫運送 我們培養後再自行抽取RNA 比較不佔用您的助理太多時間

再次感謝老師的幫忙 敬祝

研安

蔡佳龍敬上 桃園市龜山區復興街五號

林口長庚醫院兒童醫療大樓J棟3樓婦產部實驗室 蔡佳龍
電話:03­3281200­8738或8784

[Quoted text hidden]

Su­Fang Lin Fri, Jan 30, 2015 at 5:10 PM To: 蔡佳龍
Cc: Angel Chao , 王子豪醫師

Dear 佳龍:

因為我們實驗室主要是做頭頸癌, SW780膀胱癌細胞當初是養幾代抽完RNA後就 沒有再maintain, 所以送細胞和送RNA是一樣得用乾冰的. 另外是 SW780我是和人 要來的, 得要再寫信去國外友人處問可不可以分讓,有MTA問題, 反而比較麻煩。

乾冰運送RNA我有經驗、事實上半年前高醫有人跟我要SW780 RNA,目的與你們 一樣,也是當positive control, in which the results looked good. 所以我才有信心 跟趙醫師說沒問題.倒是FGFR3的fusion partner很多、即便同一個partner 斷點 也不同, 因此若欲以RT­PCR 方式篩選的話, 這點要小心.

Best,
Su­Fang
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­-------------------------

蔡佳龍
To: Su­Fang Lin
Cc: Angel Chao , 王子豪醫師

林老師您好: 謝謝的您幫忙與提醒

再麻煩老師了 謝謝您

佳龍敬上 

---------

Su­Fang Lin Tue, Feb 3, 2015 at 2:48 PM To: 蔡佳龍
Cc: 英潔YCK , 玉蓮YLC

Dear 佳龍,
一切順利的話我們應該明天星期三就可寄出冷凍包裹, 我們會請黑貓宅急便送, 標

示貨到付款, 寄到 桃園市龜山區復興街五號

林口長庚醫院兒童醫療大樓J棟3樓婦產部實驗室 這樣子OK嗎? 對了,可能還需要填你們實驗室電話. 謝謝!

Best, Su­Fang 

---------------------

蔡佳龍 Tue, Feb 3, 2015 at 2:55 PM To: Su­Fang Lin
Cc: 王子豪醫師 , 趙安琪醫師

林老師您好: 我們實驗室的電話

03­3281200­8784或8738 我的手機0975510816 再次感謝您的幫忙

佳龍敬上

---------------

蔡佳龍
To: Su­Fang Lin Cc: 王子豪醫師

Wed, Feb 4, 2015 at 7:27 AM

林老師您好: 我本身是在長庚醫院基因體核心實驗室工作,主要以affymetrix晶片研究基因體

的變化。
目前affymetrix新晶片human transcriptome array (HTA)號稱可以偵測alternative splicing與fusion gene。不知能否跟老師多要些sw780 RNA(10ug)讓我們可以測 試晶片能力,我們也會將晶片資料與老師分享。
再次麻煩老師

謝謝您的幫忙 佳龍敬上

----------

Su­Fang Lin Wed, Feb 4, 2015 at 8:45 AM To: 蔡佳龍 , 王子豪醫師
Bcc: 玉蓮YLC

Dear 佳龍,

其它cell line的RNA可以嗎? 昨天我們有多分出一管OSCC cell line TW2.6的RNA (10 ug 多一點點). TW2.6也有fusion gene, 但不是FGFR3, 而是一個receptor protein tyrosine phosphatase.

因為我們習慣讓分裝好的RNA過夜冷凍, 隔天再寄. 所以要給你們的SW780 RNA昨 天已分裝好冰回. Ten­ug 分一點過去做FGFR3的RT­PCR, 剩下的做HTA應該很足 夠喔! How do you think?

Su­Fang 

-----------------------

王子豪 Reply­To: 王子豪

Dear Dr. Lin:

Wed, Feb 4, 2015 at 9:20 AM

Thanks for the reply. If you permit, we will analyze the SW780 RNA with HTA microarrays too and report the results to you as soon as we get them.

Best regards, Tzu­Hao 

---------------------------------

Su­Fang Lin Wed, Feb 4, 2015 at 9:36 AM To: 王子豪
Cc: 蔡佳龍
Bcc: 英潔YCK , 玉蓮YLC

Dear Dr. Wang,
Are you talking about MTA thing? 據我所知, SW780的 gene fusion至少有兩篇

paper都有發表了, 所以我覺得OK.

上週佳龍問我要cells時,那就會有concern,因為我也是跟友人要來的,要分讓給 你們, 我的習慣是還會請你們跟他簽一次MTA. 想說如果只是做control, 給RNA就 可避掉這些麻煩啊.

Thus, go ahead and use both TW2.6 and SW780 RNA for your HTA analysis. 各 10­ug 應該夠吧, 還是你們需要更多, 那我們就要再回溶一次SW780, 延遲到明天 寄 ­­> Which is also fine with us.

Best, Su­Fang 

------------------

柯英潔 Wed, Feb 4, 2015 at 4:35 PM To: "hcchen@actgenomics.com"
Cc: 林素芳 , 陳玉蓮

陳博士您好

今天下午已經將RNA樣本用黑貓冷凍寄送給您 預計明天中午前會收到 請依照以下步驟沉澱RNA使用

每管約為12ug RNA溶於formamide,體積20ul,根據以往經驗沉澱後回收約80% 以上,因此最後會得到約10ug的RNA。
沉澱步驟為
1. add 1/10V 3M sodium acetate

2. add 3V EtOH(Merck), mix well 3. ­20°C O/N
4. 14500rpm, 15min
5. 70% EtOH(cold) 100ul wash 6. 14500rpm, 6min

7. Dry pellet
8. Resuspend in RNase­free H2O 9. Nanodrop

 如有任何問題再跟我聯繫
 謝謝

柯英潔 分機35122 林素芳老師實驗室 癌症研究所 國家衛生研究院 

----------------

Hua Chien Chen (陳華鍵)

To: 柯英潔
Cc: 林素芳 , 陳玉蓮 , "Bernett Hsu (徐安)"

Dear 英潔
謝謝你寄來的 RNA 以及後續處理相關資訊,我會請公司同仁依此實驗流程操

Wed, Feb 4, 2015 at 4:45 PM

作。謝謝!

陳華鍵

[Quoted text hidden]

This e­mail may contain confidential information of ACT Genomics Co., Ltd. If you are not the addressee, please notify the sender and delete this e­mail immediately. Any unauthorized review, use or distribution by anyone other than the intended recipient is strictly prohibited.

柯英潔 Wed, Feb 4, 2015 at 4:48 PM To: "Hua Chien Chen (陳華鍵)"
Cc: 林素芳 , 陳玉蓮 , "Bernett Hsu (徐安)"

陳老師您好

我想再補充二點
1. RNA在formamide中很穩定,如果還沒要往下做,直接丟 ­80C冰箱冷凍起來。 2. 步驟中,­20度沉澱只需要1小時以上即可

柯英潔 分機35122
林素芳老師實驗室
癌症研究所
國家衛生研究院 

----------------------

Hua Chien Chen (陳華鍵)

To: 柯英潔
Cc: 林素芳 , 陳玉蓮 , "Bernett Hsu (徐安)"

Dear 英潔
寄來的 RNA sample 已收到了 謝謝!

陳華鍵 

------------------

Oligo檔案區

目前想到的方法如下，請大家集思廣益讓「編號引子」成為正確有效又快樂之工作! 謝謝大家~

Step 1: 拉到本文下方第一則回應、看看最新一個人引子的LAB ID是用到第幾號了, 記住你要開始的號碼.

Step 2: 利用本文右下方「增加新的回應」

Step 3: 主題部分標入你login時間及大名 

Step 4: 回去你的Excel檔做編輯

**Excel檔來自生工 : (1) 在最前面加一欄 LAB ID (2) 輸入剛剛記住的號碼並完成編號 (3) copy A to G的資料 paste在ELN回應內文. (4) 記住設定table大小為700 (px) (範例見下面2015/01/01小嬿)

**Excel檔來自每得IDT : (1) 在最前面加一欄 LAB ID (2) 輸入剛剛記住的號碼並完成編號 (3) Move "Tm"、"Final nmoles"、"H₂O to 100 uM"、"product bp" up to "Sequence". copy A to H的資料 paste在ELN回應內文. (4) 記住設定table大小為700 (px) (範例見下面2015/01/23 Ingrid)

建議大家自己有一個Excel檔，專門存放oligo sequences..

ps. 範例2015/01/01 小嬿那一筆的LAB ID是瞎編的，最後會刪掉; 2015/01/23 Ingrid這一筆是真的，請下一個幸運兒從978開始使用! Thanks.

            

2015-02-10 Journal Club Paper

Dear all:

這是我下週要報告的paper

題目為:Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration

敬請多多指教

謝謝

    思涵 敬上

Fusion gene detection 2014-Dec 及 TLCN檢體申請

Fusion gene detection 2014-Dec 及 TLCN檢體申請
由 sufang 在 三, 01/21/2015 - 11:29 發表 Cholangiocarcinoma cholangiocarcinoma FGFR2 gene fusion

Nov 27, 2014 at 2:57 PM

<910208 mail.kmuh.org.tw=""> To: Su-Fang Lin

Dear SF,

Only minor suggestions (in red color). Are you going to list the oncogenes screening in your proposal or we can just perform the exam?

I am current reading most recent driver mutations in the cholabgiocarcinoma, but I will be great appreciated if you can also give me the most updated/recurrent mutated genes in cholangiocarcinoma. Than I will design primers for those candidate genes.

Furthermore, I do not have a MiSeq in my lab yet, so I outsourcing now. I wish

I can get my own MiSeq machine in one year.

Kindly regards, Daw-yang

Thu, Nov 27, 2014 at 4:34 PM

Su-Fang Lin To: Daw-yang Hwang

Three references for you! Best, Su-Fang

4001_2014_03_New Routes to Targeted Therapy of Intrahepatic Cholangiocarcinomas Revealed by Next-Generatiion Sequencing.pdf

4239_2013_12_Exome sequencing identifies distinct mutational patterns in liver fluke-related and non-infection- related bile duct cancers.pdf

3970_2013_12_Exome sequencing identifies frequent inactivating mutations in BAP1, ARID1A and PBRM1 in intrahepatic cholangiocarcinomas.pdf

Daw-yang Hwang <910208 mail.kmuh.org.tw=""> To: Su-Fang Lin

Please see the attached file for the updated TLCN form. Daw-yang

about your application

6 messages

To: "sflin1@gmail.com"

Dear Dr.Lin,

Mon, Dec 1, 2014 at 7:22 PM

Welcome to apply for TLCN samples. However, I need to tell you that many of the cholangiocarcinoma samples are not so good in the RNA quality (due to tumor necrosis), and many have low tumor %. Thus, though we have more than 150 cases, but it is impossible to get 96 cholangiocarcinoma with high tumor % and good RNA quality for your application. we probalbly can give you around 40-50 cases. If you just need DNA and tissue section, it may be possible.

shiufeng

Su-Fang Lin

To: <910208 mail.kmuh.org.tw="">

Dear Daw-Yang,

Mon, Dec 1, 2014 at 9:05 PM

What do you think? Can I tell her that for our purpose not so ok RNA are still worth a try? Thank you very much! SF

Sent from Gmail Mobile

Daw-yang Hwang <910208 mail.kmuh.org.tw=""> To: Su-Fang Lin

Dear SF,

Tue, Dec 2, 2014 at 5:06 AM

I can understand the RNA quality issue. We can try 48 RNA samples first to see how it goes. For DNA, it can be as much as possible, so maybe we should apply 144 DNA samples?

Daw-yang

Su-Fang Lin Tue, Dec 2, 2014 at 9:37 AM To: ,

Dear Dr. Huang,

Thank you very much for telling us the tumor necrosis/poor RNA quality issue. With this regard, we would like to apply 48 RNA samples and associated unstained paraffin sections; and 144 DNA samples of cholangiocarcinoma tissues. A new application form with revised case numbers is attached in the mail.

Thank you very much for your great help!!

Best, Su-Fang

TLCN sample_request_NICR_LinSF v2.pdf 282K

To: Su-Fang Lin ,

144 is too many. Please reduce to 80 to 90 cases.

Do you accept cases with a tumor percentage around 50 percent only? iPhone 

Su-Fang Lin To:  

Tue, Dec 2, 2014 at 12:25 PM

114太多啊! 真對不起，因為機器一次跑48個所以想說取它的倍數來申請...

好，那我們就申請九十例DNA檢體。另外，50% tumor cellularity 應該是OK的。謝謝妳!

素芳

---

Re: about your application

9 messages

 Sat, Dec 6, 2014 at 9:10 PM 

To: Su-Fang Lin Cc:

Please remember to submit a revised form of LOI.

Su-Fang Lin Sat, Dec 6, 2014 at 10:19 PM
To: ,
Dear Dr. Huang,
Enclosed please find my revised LOI. 

Again, many thanks for all your advice and help!

Best, Su-Fang

Letter of Intent of TLCN_NICR_SuFangLin v3.pdf


To: Su-Fang Lin Sun, Dec 7, 2014 at 8:17 AM

You did not change the case number for DNA. Many ICC tumors have low DNA tumor %, and need to use laser capture microdissection, which will be quite expenstive (1000/ 1ug). Anyway, the total number still can not reach 144 cases. Could you reduced to 96 cases (48 wells x2) for DNA?

shiufeng 

Su-Fang Lin To:  Sun, Dec 7, 2014 at 10:00 AM

咦，我在昨天LOI (v3)有改成90例DNA檢體及50% tumor cellularity. 是還有地方漏掉、沒改到的嗎、還是檔案沒有附檔成功? 讓我再送一次! 麻煩您再檢查一下，不好意思。

Best,

Su-Fang

sfhuang@nhri.org.tw To: Su-Fang Lin Sun, Dec 7, 2014 at 8:42 PM

You only made change in the Item V, but you did not change in Item III, No.2, which is the most important part. I can not revised the LOI for you, since it is in pdf file.

shiufeng

Su-Fang Lin Sun, Dec 7, 2014 at 9:03 PM To:

真是糊塗，不好意思. Best, Su-Fang

sfhuang@nhri.org.tw  Sun, Dec 7, 2014 at 9:11 PM

To: Su-Fang Lin

OK. It will be sent out for review.

Shiufeng

Su-Fang Lin To:

Great, thank you very much, Dr. Huang!

sfhuang@nhri.org.tw To: Su-Fang Lin Sun, Dec 7, 2014 at 9:19 PM

you are welcome.

Shiufeng

#1由 sufang 在 三, 01/21/2015 - 15:23 發表。
Regarding sample request at TLCN