Notebook

Larval Transcriptome of OsHV exposed oysters¶

A re-examination of Cgigas larval transcriptome using genome.

Solid Files -- http://eagle.fish.washington.edu/whale/index.php?dir=GE%2Freads%2F

Importing into CLCv6

Quality trim¶

Ambiguous trim = Yes Ambiguous limit = 2 Quality trim = Yes Quality limit = 0.05 Create report = Yes Save discarded sequences = No Remove 5' terminal nucleotides = No Minimum number of nucleotides in reads = 20 Discard short reads = Yes Remove 3' terminal nucleotides = No Discard long reads = No Save broken pairs = No

QC Report¶

https://docs.google.com/file/d/0B9V_gF766XZAQXp4OE45VXc0U00/preview

Mapping to Transcriptome¶

RNA-seq to genes

Parameters

Use strand specific assembly = No Create report = Yes References = oyster_v9_gene Count paired reads as two = No Use colorspace encoding = Yes Minimum number of reads = 10 Additional upstream bases = 0 Minimum read count fusion gene table = 5 Minimum length of putative exons = 25 Minimum exon coverage fraction = 0.2 Minimum length fraction (long reads) = 0.9 Use annotations for gene and transcript identification = No Create fusion gene table = No Expression value = TOTAL_GENE_COUNT Minimum similarity fraction = 0.8 Expression level = Genes Create list of unmapped reads = No Unspecific match limit = 5 Exon discovery = No Organism type = PROKARYOTE Additional downstream bases = 0 Maximum paired distance = 250 Minimum paired distance = 180 Strand = Forward Maximum number of mismatches allowed (applies to short reads) = 2 Expression value = Number of reads mapped to the gene

In [3]:

#tab delim RNA-Seq file
!head /Volumes/web/cnidarian/solid0078_20091105_RobertsLab_GE_F3\ trimmed\ RNA-Seq.txt

"Feature ID"	"Expression values"	"Gene length"	"Unique gene reads"	"Total gene reads"	"RPKM"
CGI_10000780	2	1350	2	2	0.272
CGI_10000456	28	438	18	28	11.735
CGI_10000457	5	603	4	5	1.522
CGI_10000774	1	375	1	1	0.49
CGI_10000917	2	426	2	2	0.862
CGI_10000861	7	2004	7	7	0.641
CGI_10000994	96	1635	74	96	10.779
CGI_10000643	0	552	0	0	0
CGI_10000763	4	249	4	4	2.949

In [5]:

#SAM output
!head /Volumes/web/cnidarian/solid0078_20091105_RobertsLab_GE_F3_tr_RNA-Seq.sam

@HD	VN:1.0	SO:unsorted
@SQ	SN:CGI_10000780	LN:1350
@SQ	SN:CGI_10000456	LN:438
@SQ	SN:CGI_10000457	LN:603
@SQ	SN:CGI_10000774	LN:375
@SQ	SN:CGI_10000917	LN:426
@SQ	SN:CGI_10000861	LN:2004
@SQ	SN:CGI_10000994	LN:1635
@SQ	SN:CGI_10000643	LN:552
@SQ	SN:CGI_10000763	LN:249

In [8]:

#SAM output
!tail /Volumes/web/cnidarian/solid0078_20091105_RobertsLab_GE_F3_tr_RNA-Seq.sam

read_5447380	0	CGI_10003117	263	60	50M	*	0	0	CCTCGCATTGGAAAACCCCAGTGTTTGGGTGGGTGTCACAAGAGGAAGAA	@A@?B=AA;@BB@:@<BA?:?B=:92?3;@?>:;<=2;>6:?98>:77/7	NH:i:1	CS:Z:T20223313010200010001211100100110011121110222020220
read_5447381	0	CGI_10003117	357	60	48M	*	0	0	ACCATTGAATCGATTTCCAAATGTGATGTTCCATCTGCAGTCCCTCTT	B@B@BA?ABBAAABAABBA@5A@?@ABA>AAB@=?AB@?@7@@8@?@5	NH:i:1	CS:Z:T310130120323230020100311123110201322131212002220
read_5447382	0	CGI_10003117	375	60	44M	*	0	0	AAATGTGATGTTCCATCTGCAGTCCCTCTTTAAATGGTCACAGT	@?BB@@BABB>B@A??BBB9=B:B@@AAB<59>@><@>A?>?<:	NH:i:1	CS:Z:T30031112311020132213121200222003003101211121
read_5447383	0	CGI_10003117	375	60	44M	*	0	0	AAATGTGATGTTCCATCTGCAGTCCCTCTTTAAATGGTCACAGT	>@BB@?BBBA<BAA=ABAB<=@;>>@B>@<78?@=9@?A>>?;;	NH:i:1	CS:Z:T30031112311020132213121200222003003101211121
read_5447384	0	CGI_10003117	375	60	44M	*	0	0	AAATGTGATGTTCCATCTGCAGTCCCTCTTTAAATGGTCACAGT	@BB@@>@BAA=@@B?=B@B::@>5==?@@=/*?B81>;AA?@:7	NH:i:1	CS:Z:T30031112311020132213121200222003003101211121
read_5447385	0	CGI_10003117	507	60	29M	*	0	0	GATGAATTGGTATAACATTGTCAACTCTT	BBB?<@BBA=@BA?>=AA@;6@?<:8AA/	NH:i:1	CS:Z:T12312030101333011301121012220
read_5447386	0	CGI_10003117	609	60	3S35M	*	0	0	AGCACAGATCCAAACTGGAATTTACCAGAACCGCCAGA	BBBABBBABBBB>BA>@??BB@?AB@B<3@A>=9AA>=	NH:i:1	CS:Z:T32311122320100121020300310122010330122
read_5447387	0	CGI_10003117	609	60	3S47M	*	0	0	AGCACAAATCCAAACTGGAATTTACCAGAACCGCCAGAAGAATACATCCC	BBBABAA=BBBA@?B@=@?AB??BA?@<;A@:>=@>8:7@=><=@=<5;?	NH:i:1	CS:Z:T32311100320100121020300310122010330122022033113200
read_5447388	0	CGI_10003117	609	60	3S35M	*	0	0	AGCACAGATCCAAACTGGAATTTACCAGAACCGCCAGA	BBBBBBB@BBBBBB@B?BBBBB@BB@@==BA;A>AA@=	NH:i:1	CS:Z:T32311122320100121020300310122010330122
read_5447389	0	CGI_10003117	609	60	48M2S	*	0	0	ACAGATCCAAACTGGAATTTACCAGAACCGCCAGAAGAATACATCCCCCC	BA@BBBBAB@BBAAB>ABB?BAA??>;=??>A>=97<?>@@AA537<;@=	NH:i:1	CS:Z:T31122320100121020300310122010330122022033113200000

In [39]:

#Lets figure out a way to visualize what genes are expressed
#Take RNA-seq file import into SQLShare
#imported /Volumes/web/cnidarian/solid0078_20091105_RobertsLab_GE_F3\ trimmed\ RNA-Seq.txt
#cleaned up

https://sqlshare.escience.washington.edu/sqlshare#s=query/sr320%40washington.edu/solid0078_20091105_RobertsLab_GE_F3%20trimmed%20RNA-Seq.txt

In [42]:

#joining with annotation data.
#create generic SQLShare Wiki workflow.

https://github.com/uwescience/sqlshare/wiki/Workflow%3A-Annotating-Oyster-Genes

http://eagle.fish.washington.edu/cnidarian/OsHV_larvae_RNAseq_UR10.txt

In [43]:

!head /Volumes/web/cnidarian/Cgigas_larvae_RNAseq_OsHV_GO.csv

In [44]:

!wc /Volumes/web/cnidarian/Cgigas_larvae_RNAseq_OsHV_GO.csv

  121979  315335 6935565 /Volumes/web/cnidarian/Cgigas_larvae_RNAseq_OsHV_GO.csv

In [45]:

#into GoCategorizer then ManyEyes

Many%20Eyes%20:%20Customizing%20Bubble%20Chart

Enrichment Analysis¶

Background - all SPIDs associated with oyster transcriptome Gene list - SPID of gene with at least 10 unique reads

Kegg

DAVID:%20Database%20for%20Annotation,%20Visualization,%20and%20Integrated%20Discovery%20(Laboratory%20of%20Immunopathogenesis%20and%20Bioinformatics%20(LIB);%20National%20Institute%20of%20Allergies%20and%20Infectious%20Diseases%20(NIAID);%20Science%20Applications%20International%20Corporation%20(SAIC)

http://eagle.fish.washington.edu/cnidarian/chart_5EB4CB21C87A1374501610998.txt

BP-Fat

http://eagle.fish.washington.edu/cnidarian/chart_5EB4CB21C87A1374501882704.txt

pvalue <.05 into Revigo

GO:0008104 1.87E-10 GO:0045184 4.06E-10 GO:0015031 1.55E-09 GO:0051056 2.29E-08 GO:0046578 7.92E-07 GO:0046907 1.69E-06 GO:0006796 8.93E-06 GO:0016310 9.71E-06 GO:0006793 1.68E-05 GO:0006396 1.80E-05 GO:0016071 2.05E-05 GO:0006886 9.38E-05 GO:0043087 3.10E-04 GO:0006468 3.15E-04 GO:0034613 3.40E-04 GO:0006511 4.70E-04 GO:0006397 4.72E-04 GO:0070727 4.87E-04 GO:0006091 5.48E-04 GO:0008380 0.001634341 GO:0007049 0.001856279 GO:0032318 0.001879989 GO:0035023 0.002340023 GO:0046034 0.002847324 GO:0006644 0.002942199 GO:0007017 0.003300719 GO:0032313 0.003409026 GO:0032483 0.003409026 GO:0051169 0.003795828 GO:0019637 0.003832318 GO:0006913 0.004482562 GO:0006412 0.005141395 GO:0016568 0.005563156 GO:0050657 0.006346393 GO:0050658 0.006346393 GO:0051236 0.006346393 GO:0016265 0.006480115 GO:0007010 0.007213165 GO:0009141 0.007619486 GO:0008219 0.007723643 GO:0009142 0.008393563 GO:0009057 0.008477453 GO:0006818 0.008580567 GO:0043954 0.009700576 GO:0006325 0.009965083 GO:0009205 0.010315383 GO:0009199 0.010315383 GO:0009145 0.010315719 GO:0009206 0.010315719 GO:0009201 0.010315719 GO:0044265 0.010376148 GO:0015992 0.010857381 GO:0006403 0.011334734 GO:0012501 0.012741549 GO:0006754 0.013504604 GO:0009894 0.013504604 GO:0051301 0.014127469 GO:0046486 0.01575266 GO:0006650 0.01589459 GO:0009165 0.016043426 GO:0043632 0.016123335 GO:0019941 0.016123335 GO:0034330 0.016219917 GO:0015986 0.016219917 GO:0015985 0.016219917 GO:0045494 0.016219917 GO:0022613 0.016633231 GO:0006119 0.016730696 GO:0034622 0.016780957 GO:0006915 0.016867222 GO:0009144 0.018450072 GO:0051028 0.018450072 GO:0022618 0.018899413 GO:0007265 0.02172187 GO:0015931 0.021879897 GO:0006163 0.022368298 GO:0000278 0.02349573 GO:0009150 0.024939324 GO:0016192 0.025101667 GO:0008203 0.025445341 GO:0006413 0.025494209 GO:0034504 0.025886646 GO:0046822 0.027243792 GO:0001701 0.027310746 GO:0030163 0.028215019 GO:0016042 0.028610538 GO:0048193 0.02882247 GO:0007163 0.029465914 GO:0009259 0.032187365 GO:0044257 0.032802252 GO:0017038 0.033241064 GO:0006164 0.0341163 GO:0006605 0.034586659 GO:0051603 0.034854969 GO:0006401 0.035288451 GO:0030031 0.035977875 GO:0034621 0.038678526 GO:0055085 0.040014925 GO:0007018 0.042241787 GO:0051170 0.042255571 GO:0009311 0.042515336 GO:0016579 0.044317708 GO:0070646 0.044317708 GO:0051276 0.045536962 GO:0034654 0.045690801 GO:0034404 0.045690801 GO:0000226 0.046104738 GO:0000375 0.0482382 GO:0043161 0.049498849 GO:0010498 0.049498849

R script for treemap http://eagle.fish.washington.edu/cnidarian/OsHV_Cg_REVIGO_treemap.r

revigo_treemap.pdf__1_page__17E0FC3B.png mm

iPath¶

In [ ]:

Mapping to OsHV¶

http://www.ncbi.nlm.nih.gov/nuccore/NC_005881.1

imported genbank format to hav CDS, gene information..
started in CLC using same parameters..

      Minimum number of reads = 10
      Minimum exon coverage fraction = 0.2
      Additional downstream bases = 0
      Use colorspace encoding = Yes
      Create report = Yes
      Use strand specific assembly = No
      Count paired reads as two = No
      Minimum length fraction (long reads) = 0.9
      Additional upstream bases = 0
      Unspecific match limit = 5
      Expression value = RPKM
      Minimum read count fusion gene table = 5
      Create fusion gene table = No
      Minimum paired distance = 180
      Use annotations for gene and transcript identification = Yes
      Strand = Forward
      Organism type = PROKARYOTE
      Maximum number of mismatches allowed (applies to short reads) = 2
      Minimum similarity fraction = 0.8
      References = NC_005881
      Expression level = Genes
      Minimum length of putative exons = 25
      Maximum paired distance = 250
      Create list of unmapped reads = No
      Exon discovery = No
      Expression value = Read Per Kilobase of exon Model value

Found: 127 genes. Total number of reads : 21344598 ( single reads: 21344598, paired reads: 0 ) Total number of mapped reads : 21135 ( single reads: 21135, paired reads: 0 ) Total number of unmapped reads : 21323463 ( single reads: 21323463, paired reads: 0 )

In [2]:

!head /Volumes/web/cnidarian/solid0078_20091105_RobertsLab_GE_F3trim_RNAseqOSHV.csv

"Feature ID","Expression values","Gene length","Unique gene reads","Total gene reads","RPKM","Chromosome region start","Chromosome region end"
"OsHV1_gp001","423.399","447","0","4","423.399","115","561"
"OsHV1_gp002","751.03","504","0","8","751.03","680","1183"
"OsHV1_gp003","61.85","765","0","1","61.85","1890","2654"
"OsHV1_gp004","2748.769","1050","0","61","2748.769","3384","4433"
"OsHV1_gp005","3168.303","2031","70","136","3168.303","6421","8451"
"OsHV1_gp006","1534.465","3546","115","115","1534.465","8628","12173"
"OsHV1_gp007","1429.304","960","29","29","1429.304","12211","13170"
"OsHV1_gp008","4016.047","1779","151","151","4016.047","13258","15036"
"OsHV1_gp009","6345.436","1029","138","138","6345.436","15297","16325"

Reference Mapping to OsHV¶

SAM file http://eagle.fish.washington.edu/cnidarian/solid0078_20091105_RbbertsLab_GE_F3%20trimmed%20mapping.sam

SNP ID based on Reference Mapping¶

In [3]:

!cat /Volumes/web/cnidarian/OsHV_snp_table

"Reference Position"	"Type"	"Length"	"Reference"	"Allele"	"Linkage"	"Zygosity"	"Count"	"Coverage"	"Frequency"	"Forward/reverse balance"	"Average quality"	"Overlapping annotations"	"Coding region change"	"Amino acid change"
32003	SNV	1	G	A		Homozygous	12	12	100	0.417	23.833	Gene: OsHV1_gp021, CDS: OsHV1_gp021, Misc. feature: UL	YP_024567.1:c.1533G>A	YP_024567.1:p.Met511Ile
57377	SNV	1	C	T		Homozygous	10	10	100	0.1	24.1	Gene: OsHV1_gp039, CDS: OsHV1_gp039, Misc. feature: UL	YP_024585.1:c.1468C>T	YP_024585.1:p.Pro490Ser
83889	SNV	1	A	G		Homozygous	18	18	100	0.056	27.222	Gene: OsHV1_gp053, CDS: OsHV1_gp053, Misc. feature: UL	YP_024599.1:c.554A>G	YP_024599.1:p.Glu185Gly
92644	SNV	1	A	G		Homozygous	13	13	100	0.462	23.769	Gene: OsHV1_gp058, CDS: OsHV1_gp058, Misc. feature: UL	YP_024604.1:c.1349A>G	YP_024604.1:p.Tyr450Cys
124796	SNV	1	G	A		Homozygous	10	10	100	0.1	30.2	Gene: OsHV1_gp071, CDS: OsHV1_gp071, Misc. feature: UL	YP_024617.1:c.2083G>A	YP_024617.1:p.Ala695Thr

IGV Browser for OsHV¶

In [6]:

!head /Volumes/web/cnidarian/solid0078_20091105_RbbertsLab_GE_F3\ trimmed\ mapping.sam

@HD	VN:1.0	SO:unsorted
@SQ	SN:NC_005881	LN:207439	SP:Ostreid herpesvirus 1
@PG	ID:0	PN:clcgenomicswb	VN:7.0
read_1	0	NC_005881	2764	60	2S18M	*	0	0	CATGGGGGGGGGGGGGGGGG	BB@??979/12.=<+9-;22	NH:i:1	CS:Z:T21312000000000000000
read_2	16	NC_005881	7624	60	32M	*	0	0	CACTTGACAACATCCATACATATCGTCATCCA	>9;3,@5>/<@7?=8BA>B:B?A<=@AA;7B@	NH:i:1	CS:Z:T01023121323331133102311011200211
read_3	16	NC_005881	7624	60	26M	*	0	0	CACTTGACAACATCCATACATATCGT	?>;>2?5>?><AA@=ABA>BBBBABB	NH:i:1	CS:Z:T31323331133102311011210211
read_4	16	NC_005881	7638	60	46M	*	0	0	CATACATATCGTCATCCAGGGTCTCTCCGTGTTTAGGATATAGCGT	<?A>0=??6&A8:?9>>>@9??>@@B6@ABBA?ABBBB>BBBBBAB	NH:i:1	CS:Z:T3133233332023001113022222100210231211233311331
read_5	0	NC_005881	7640	60	34M	*	0	0	TACATATCGTCATCCAGGGTCTCTCCGTGTTTAG	BBAABBBBBABBBBABBBBAB@BB@BBA@+>BBB	NH:i:1	CS:Z:T0311333231213201200122222031110032
read_6	16	NC_005881	7640	60	44M	*	0	0	TACATATCGTCATCCAGGGTCTCTCCGTGTTTAGGATATAGCGT	=26?+:0/:04;=87232=1-1<A:<:@=@8=@BAA<@@1BA=B	NH:i:1	CS:Z:T31332333320230011130222221002102312132323113
read_7	0	NC_005881	7640	60	46M3S	*	0	0	TACATATCGTCATCCAGGGTCTCTCCGTGTTTAGGATATAGCGTATATA	BBBBBBBBB=BBBBBABB@AB@BA@BB=B/>ABA+@BB?%B@B;??6B>	NH:i:1	CS:Z:T0311333231213201200122222031110032023333233133333

In [7]:

!head /Volumes/web/cnidarian/NC_005881

>NC_005881 Ostreid herpesvirus 1, complete genome.
CCCCCCACCTCCCCAACACCTCCCCCATCCTCCCCACCTCCTCCCCCTCCTCTCTTCCGC
CCGCGATCCCGCCAATACCCATAATGCACCTGGGCACTCTCTTTTTTCCTTTCCTTATCC
AAGATGTCCGCCCATTGCCAGGTACAGCCTTCCCACCGTGTGAACAATGTCCATCCTCTT
CTCCGAGACTTCCCTGACCAGATTGTCGTAATCCAATTGACACATTCTCGTCAATGCCCT
CCTCATATTCTCCATCGGCCAACTGTCGTCTCTACTCATGGTCATAAACAATCCCAATCC
ACTCTTGGCATCCCGCAACCTTTCCAATAGCCTCCCGAATTCGTCTACCGCCGCCTTATC
GTCGTCCGTGCTGCAATGTGGTCTTACCGCATTTTTAAGCAATGCGCACGCCACTCTCAA
TTCCTGACAGGTAATCTCCTCCACCGGTTTCCTATCGTGTAATAGACTGACCACGGCGGC
CATGTCTCTCAGTTCCTTGCTCATCTCACCACCGCCAACCAATTCAGCAGTGACAGTTAC

IGV_and_Directory_Listing_of__cnidarian__and_CLC_Genomics_Workbench_7_0_4_195B0EAF.png

LINKOUT to xml http://eagle.fish.washington.edu/cnidarian/OsHV_igv_session.xml

Mapping to GQ153938¶

In [ ]: