Danio rerio Fugu rubripes Gasterosteus aculeatus Oryzias latipes Tetraodon nigroviridis Total unique 17377 2411 7747 8037 3674 24092 13.61 1.89 6.07 6.29 2.88 18.86 3．结果文件及说明
3.1 软件参数设置
以下仅列出分析所涉及到的软件参数设置，自写程序由于不涉及到任何参数及版权问题故不公开。
a. Seqclean (默认参数)
b. Lucy：-m 50 –e 0.03 0.03 –w 30 0.03 10 0.1 –b 4 0.03
参数 -m -e[1] -e[2] -w[1] -w[2] -w[3] -w[4] -b[1] -b[2] 说明 序列最短长度(bp) 平均最大错误率 序列尾部最大错误率 滑动窗口长度(bp) 窗口内平均最大错误率 大滑动窗口内小滑动窗长度(bp) 窗口内平均最大错误率 序列头、尾检验长度(bp) 最大错误率 参数设置 50 0.03 0.03 30 0.03 10 0.1 4 0.03 c. Blastx：-m 0 –F F –e 1e-3 –b 5 –v 5
参数 -m -F
说明 输出格式 低复杂度序列是否过滤 参数设置 0 F 10
-e -b -v d. Newbler
参数 Use duplicate reads Extend low depth overlaps Minimus read length Reads limited to one contig 说明 拼接过程是否使用duplicate序列 低Depth区域是否延伸 Reads最短长度(bp) [20-45] 一个Reads是否只允许出现在一条Contig中 参数设置 Checked Checked 45 Checked 期望值 输出alignment的序列个数 输出 1e-3 5 5 e. Misa：1-10, 2-6, 3-5, 4-5, 5-5, 6-5, max_difference_between_2_SSRs=100
参数 1 2 3 4 5 6 Max_difference_between_2_SSRs 说明 单碱基连续重复次数 双碱基连续重复次数 3碱基连续重复次数 4碱基连续重复次数 5碱基连续重复次数 6碱基连续重复次数 两个SSR间最大间隔长度(bp) 参数设置 10 6 5 5 5 5 100 3.2 结果文件目录说明
目录 /assembly_result /database_annotation /SSR /SNP /GO /different_gene 说明 Newbler拼接结果文件 Swiss-Prot注释后结果文件 SSR分析结果文件 SNP分析结果文件 GO分析结果文件 差异基因分析结果 11
/tblastx Unigene与EST/mRNA比对结果 4. 参考文献
[1] Camacho C., Coulouris G., Avagyan V., Ma N., Papadopoulos J., Bealer K., & Madden T.L. (2008) \
[2] http://sourceforge.net/projects/seqclean/
[3] LUCY2: an interactive DNA sequence quality trimming and vector removal tool Bioinformatics Advance Access published on May 6, 2004
[4] Huang, X. and Madan, A. (1999) CAP3: A DNA Sequence Assembly Program, Genome Research, 9: 868-877.
[5] http://pgrc.ipk-gatersleben.de/misa/
[6] ssahaSNP A Polymorphism Detection Tool on a Whole Genome Scale 2005 IEEE Computational Systems Bioinformatics Conference - Workshops, Vol. 0 (2005), pp. 251-254.
[7] Gene ontology: tool for the unification of biology. Nat. Genet.. May 2000;25(1):25-9. [8] Kanehisa, M., Goto, S., Furumichi, M., Tanabe, M., and Hirakawa, M.; KEGG for representation and analysis of molecular networks involving diseases and drugs. Nucleic Acids Res. 38, D355-D360 (2010).
[9] The Universal Protein Resource (UniProt) in 2010 Nucleic Acids Res. 38:D142-D148 (2010).
5. 附录（实验部分protocol）
5.1 RNA提取
*Invitrogen TRIzol Reagent
5.1.1 RNA precipitation
a.
(Optional) When precipitating RNA from small sample quantities (<106 cells or <10 mg tissue), add
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5–10 μg of RNase-free glycogen as a carrier to the aqueous phase. b.
Note: Glycogen is co-precipitated with the RNA, but does not inhibit first-strand synthesis at concentrations ≤4 mg/mL, and does not inhibit PCR. c.
Add 0.5 mL of 100% isopropanol to the aqueous phase, per 1 mL of TRIzol? Reagent used for homogenization. d. e. f.
Incubate at room temperature for 10 minutes. Centrifuge at 12,000 × g for 10 minutes at 4°C.
Note: The RNA is often invisible prior to centrifugation, and forms a gel-like pellet on the side and bottom of the tube. g.
Proceed to RNA wash.
5.1.2 RNA wash
a. b.
Remove the supernatant from the tube, leaving only the RNA pellet.
Wash the pellet, with 1 mL of 75% ethanol per 1 mL of TRIzol? Reagent used in the initial homogenization. c. d. e. f.
Note: The RNA can be stored in 75% ethanol at least 1 year at –20°C, or at least 1 week at 4°C. Vortex the sample briefly, then centrifuge the tube at 7500 × g for 5 minutes at 4°C. Discard the wash. Vacuum or air dry the RNA pellet for 5–10 minutes. Do not dry the pellet by vacuum centrifuge. Note: Do not allow the RNA to dry completely, because the pellet can lose solubility. Partially dissolved RNA samples have an A260/280 ratio <1.6. g.
Proceed to RNA resuspension.
5.1.3 RNA resuspension
a.
Resuspend the RNA pellet in RNase-free water or 0.5% SDS solution (20–50 μL) by passing the solution up and down several times through a pipette tip. b. c.
Note: Do not dissolve the RNA in 0.5% SDS if it is to be used in subsequent enzymatic reactions. Incubate in a water bath or heat block set at 55–60°C for 10–15 minutes. Proceed to downstream application, or store at –70°C.
d.
5.2反转录成cDNA
*Clonetech SMART
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5.2.1 First-strand cDNA synthesis, set up 2 tubes for 2ug polyA RNA, for each tube:
a.
add the following components to a nuclease-free 0.2ml PCR tube:
X ul Nuclease-free H2O 1-10ul
1ug polyA RNA
1ul dT15VN2 primer (50uM) 1ul 10mM dNTP
----------------------------------------------- 12ul Total
b. briefly mix, spin.. c. 65C, 5min. 4C 2min.
d.
add the following components: 4ul 5x First-Strand buffer 1ul 0.1M DTT 1ul RNaseOUT (40u/ul) 2ul
SuperScript III RT (200u/ul)
--------------------------------------------------- 20ul Total e. 50C, 1h f.
70C, 15min
g. 4C, 2 min, spin the content down
5.2.2 Second strand synthesis, for each tube:
a.
add the following component:
91ul Nuclease-free H2O 30ul 5X Second-Strand buffer 3ul 10mM dNTP
1ul E coli DNA ligase(10U/ul) 4ul E coli DNA polymerase(10U/ul) 1ul
E coli RNaseH (2 U/ul)
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