De novo transcriptome sequencing and analysis of Anisakis pegreffii (Nematoda: Anisakidae) third-stage and fourth stage larvae

Nam, U-Hwa; Kim, Jong-Oh; Kim, Jeong-Ho

De novo transcriptome sequencing and analysis of Anisakis pegreffii (Nematoda: Anisakidae) third-stage and fourth stage larvae

Journal of Nematology

Volume 52 (2020): Issue 1 (January 2020)

By:

U-Hwa Nam, Jong-Oh Kim and Jeong-Ho Kim

Open Access

|Apr 2020

Figures & Tables

GO analysis of DEGs in A. pegreffii transcriptomes (A. pegreffii L3 vs A. pegreffii L4).

The expression level of selected DEGs either in APL3 or in APL4 by real-time PCR. A: the real-time PCR results of five genes with significantly higher expression level in APL3 compared to APL4; B: the real-time PCR results of five genes with significantly higher expression level in APL4 compared to APL3; C and D are 14 additional genes selected to compare the expression levels of ASL3 and APL4 (C: eight gene for APL3 compared to APL4, D: six gene for APL4 compared to APL3). GAPDH genes were used as the reference genes. The results are given as mean of samples (n = 3) and expressed as fold change in mRNA expression. *P < 0.05.

Top 20 most differentially expressed genes in A_ pegreffi L3_

No.	Name	Description	Log FC	P-value	Q-value
1	ergic2	Endoplasmic reticulum-Golgi intermediate compartment protein 2	16.8	0.0001179	0.247384
2	mgat4b	Alpha-1,3-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase B	16.7	0.0001274	0.247384
3	Acin1	Apoptotic chromatin condensation inducer in the nucleus	16.3	0.0002229	0.255429
4	SFXN2	Sideroflexin-2	16.1	0.0003069	0.265269
5	ANKHD1	Ankyrin repeat and KH domain-containing protein 1	16.1	0.0002901	0.265269
6	RALGAPA1	Ral GTPase-activating protein subunit alpha-1	15.9	0.0003816	0.278843
7	SI	Sucrase-isomaltase, intestinal	15.9	0.0004035	0.278843
8	B0361.11	Putative transporter B0361.11	15.9	0.0004042	0.278843
9	lact-2	Beta-lactamase domain-containing protein 2	15.8	0.0004392	0.291452
10	sptl-2	Serine palmitoyltransferase 2	15.8	0.0004306	0.291452
11	hyd	E3 ubiquitin-protein ligase hyd	15.7	0.0005016	0.292532
12	Macf1	Microtubule-actin cross-linking factor 1	15.7	0.0005216	0.292532
13	ZK370.4	Uncharacterized NTE family protein ZK370.4	15.7	0.000554	0.292532
14	LOAG_00120	Hypothetical protein	15.7	0.0005268	0.292532
15	FZD10	Frizzled-10	15.6	0.0005786	0.292532
16	cdgs-1	Phosphatidate cytidylyltransferase	15.6	0.0005679	0.292532
17	Pcsk1	Neuroendocrine convertase 1	15.6	0.000601	0.292532
18	Bm1_49625	Hypothetical protein	15.6	0.0006287	0.292532
19	GLSN_MEDSA	Glutamate synthase [NADH], amyloplastic	15.6	0.0006022	0.292532
20	Moe	Moesin/ezrin/radixin homolog 1	15.6	0.0005762	0.292532

Gene expression overview of A_ pegreffii L3 and L4 transcriptomes_

	Gene (fpkm < 1.0)
Name	Expressed	Known	Novel	Unexpressed
APL3	47,243	18,753	28,490	3,030
APL4	43,660	18,996	24,664	6,613

Preprocessing statistics of Anisakis transcriptome_

Name	Raw reads		Clean reads
	Reads	Base pairs	Reads (%)	Base pairs (%)
APL3	57,831,158	4,395,168,008	53,790,942 (93.0%)	4,079,160,122 (92.8%)
APL4	61,963,202	4,709,203,352	56,726,910 (91.5%)	4,301,151,997 (91.3%)

Top 20 most abundant unigenes in A_ pegreffii L4_

No.	ID	Name	Description
1	TBIU007721	PI-3	Major pepsin inhibitor 3
2	TBIU033076	col-145	Putative cuticle collagen 145
3	TBIU014455	col-34	Cuticle collagen 34
4	TBIU001741	col-34	Cuticle collagen 34
5	TBIU018580	ART2	Putative uncharacterized protein ART2
6	TBIU006603	COIII	Cytochrome c oxidase subunit 3
7	TBIU020343	col-138	Protein COL-138
8	TBIU020344	Bm1_54705	Nematode cuticle collagen N-terminal domain containing protein
9	TBIU013810	COI	Cytochrome c oxidase subunit 1
10	TBIU000598	col-40	Cuticle collagen 40
11	TBIU008478	COII	Cytochrome c oxidase subunit 2
12	TBIU003870	Y51F10.7	Protein Y51F10.7
13	TBIU015232	IXCI_RHIMP	Chymotrypsin-elastase inhibitor ixodidin
14	TBIU017350	ZK970.7	Protein ZK970.7
15	TBIU001757	Bm1_23600	hypothetical protein
16	TBIU015233	IXCI_RHIMP	Chymotrypsin-elastase inhibitor ixodidin
17	TBIU006107	col-182	Protein COL-182
18	TBIU029263	CRVP	Venom allergen 3
19	TBIU004632	col-34	Cuticle collagen 34
20	TBIU030958	LGALS8	Galectin-8

Top 20 most abundant unigenes in A_ pegreffii L3_

No.	ID	Name	Description
1	TBIU006603	COIII	Cytochrome c oxidase subunit 3
2	TBIU013810	COI	Cytochrome c oxidase subunit 1
3	TBIU015558	UBB	Polyubiquitin-B
4	TBIU017350	ZK970.7	Protein ZK970.7
5	TBIU017923	act-2b	Actin-2
6	TBIU015560	UBB	Polyubiquitin-B
7	TBIU017925	act-2b	Actin-2
8	TBIU008478	COII	Cytochrome c oxidase subunit 2
9	TBIU015561	UBIQP_XENLA	Polyubiquitin
10	TBIU002664	UBIQP_XENLA	Polyubiquitin
11	TBIU022916	HSP70	Heat shock 70 kDa protein
12	TBIU007173	NAP1L4	Nucleosome assembly protein 1-like 4
13	TBIU018580	ART2	Putative uncharacterized protein ART2
14	TBIU001952	R09H10.3	Probable 5-hydroxyisourate hydrolase R09H10.3
15	TBIU003351	PCCB	Propionyl-CoA carboxylase beta chain, mitochondrial
16	TBIU022918	HSP70	Heat shock 70 kDa protein
17	TBIU030005	ASP2_ANISI	Serine protease inhibitor 2
18	TBIU005085	Mmadhc	Methylmalonic aciduria and homocystinuria type D homolog, mitochondrial
19	TBIU032617	CLPH_ONCVO	Calponin homolog OV9M
20	TBIU030096	Mcee	Methylmalonyl-CoA epimerase, mitochondrial

Primer information for real-time PCR analysis_

Stage	Gene	Primer sequences (5´ − 3´)	Length (bp)
APL3	Acin1	F: 5´-TGAATGACGAAGAAGGGAGATGA-3´	108
		R: 5´-CAGTGTCAAATGAGAATAGCGTTTC-3´
	SI	F: 5´-GAGGCGATTGCTGGAAACAT-3´	111
		R: 5´-CTTCGTTGGTTCTTTTTGTCGTT-3´
	lact-2	F: 5´-CACCCCACCATCCTCTCCTT-3´	114
		R: 5´-CCGAGTGTATCTGCGAGGAAA-3´
	sptl-2	F: 5´-GGCAACCAAGAACGAGTGATG-3´	108
		R: 5´-TCAGCATGGGATTTGCAACA-3´
	Macf1	F: 5´-ATGCTTGCTCCAGTTCTCCTAAA-3´	125
		R: 5´-ATGGACAGGACGCTCTTCTTG-3´
	ergic2	F: 5´-CTGTTGAGTTGATGGCTGGAAA-3´	106
		R: 5´-ATTTTGGCACCACTGCTTTTATTAG-3´
	mgat4b	F: 5´-AATGCCAGCGATTGAAGAGTTT-3´	115
		R: 5´-CGCTGGTGTACGCAAATCATA-3´
	B036.11	F: 5´-TAATGATTGCTGAATGCGTCTGT-3´	120
		R: 5´-ACACCGAAAGATAACAACGAATACG-3´
	nas-13	F: 5´-AGCAATAGCAGCAGCGATGA-3´	131
		R: 5´-GTAGCCAATGCTTTT-3´
	EF-TSMT	F: 5´-TTCTTCTACTCCTCATCGCATCTG-3´	100
		R: 5´-TTATGCTCCTCCAGTGCTTTTG-3´
	SFXN2	F: 5´-TTAGAATGGCGTTGAAGCAGTAGTAG-3´	130
		R: 5´-AGTATCGGTTCTGACCAGTTTTTTG-3´
	hyd	F: 5´-CCATCCAGTGAAGAAGGATTCC-3´	116
		R: 5´-GAATAGAGCGGTAAGTAGAGCCTTGA-3´
	dhs-27	F: 5´-ACGATGAACGATTCGGGTAAGT-3´	140
		R: 5´-GCGTTATCCCCACATTTTCAA-3´
APL4	vps-11	F: 5´-CTCAACACACCACAATACTCATCAAT	105
		R: 5´-CAGCAACATCAACATCACAACTCA
	EGF1	F: 5´-CACATCAGCCCAGAAACATACG	106
		R: 5´-ATTTAGACCGTCGCCAGGAA
	CLINT1	F: 5´-AACGCAAGAATAGGCAAACACA-3´	113
		R: 5´-AGGAGCATCAACAGAAAACAATGAG-3´
	let-805	F: 5´-CACCGAGCACCGCTATCAA-3´	120
		R: 5´-GAAGGAGGCATCAAGGAAAGTG-3´
	VIT	F: 5´-AGGCTTATCATTACTTCGTTCACTCA-3´	105
		R: 5´-GTTTGGTTGTCGGTTCAGGTGTA-3´
	F09E10.7	F: 5´-CCACGCTCAAAAGCCCATT-3´	116
		R: 5´-GGACCAGCGGAAGTTCAGAA-3´
	ATP8B4	F: 5´-CGACACATTCTTCGTCAGCATT-3´	135
		R: 5´-CCGAACCCAAGCATGAAAA-3´
	pept-1	F: 5´-TGCTCAAGGAGAAGGAAGTTTACA-3´	113
		R: 5´-TACCCCACTTCACCGATTCG-3´
	col-34	F: 5´-GCCGAGTAAGCCACAAAACG-3´	121
		R: 5´-TACACCCCGTCCACCTTCA-3´
	col-40	F: TTCAGCAGGTGGGCAGTCA	98
		R: ATGGAACTCCGGGTGAGGAT
	SAM-MT	F: 5´-CCATCAAGGCGGCGTTAC-3´	93
		R: 5´-CAGCATGCCATAGATCCAGTGT-3´

Top 20 most differentially expressed genes in A_ pegreffi L4_

No	Name	Description	Log FC	p-value	q-value
1	F09E10.7	Protein F09E10.7	20.7	0.0000115	0.171197
2	ATP8B4	Probable phospholipid-transporting ATPase IM	18.7	0.0000172	0.171197
3	pept-1	Peptide transporter family 1	18.5	0.0000214	0.171197
4	vps-11	Vacuolar protein sorting-associated protein 11 homolog	18.5	0.0000214	0.171197
5	col-34	Cuticle collagen 34	18.4	0.0000229	0.171197
6	EGF1	Fibropellin-1	17.5	0.000063	0.206261
7	CLINT1	Clathrin interactor 1	17.4	0.0000751	0.218706
8	let-805	Protein LET-805	17.2	0.0000989	0.247384
9	Bm1_33530	Hypothetical protein	17	0.0001257	0.247384
10	VIT	Vitrin	17	0.0001194	0.247384
11	col-40	Cuticle collagen 40	16.9	0.0001524	0.255429
12	VIT	Vitrin	16.7	0.0002081	0.255429
13	unc-104	Kinesin-like protein unc-104	16.7	0.0001838	0.255429
14	SAM-MT	Probable fatty acid methyltransferase	16.7	0.000202	0.255429
15	sulp-3	Sulfate permease family protein 3	16.6	0.0002432	0.258155
16	CLINT1	Clathrin interactor 1	16.6	0.0002241	0.255429
17	Oplah	5-oxoprolinase	16.5	0.0002634	0.265269
18	VIT	Vitrin	16.4	0.0003171	0.265269
19	LOAG_09410	Hypothetical protein	16.4	0.0003238	0.265269
20	ZAN	Zonadhesin	16.4	0.0003204	0.265269

DOI: https://doi.org/10.21307/jofnem-2020-041 | Journal eISSN: 2640-396X | Journal ISSN: 0022-300X

Journal RSS Feed

Language: English

Page range: 1 - 16

Submitted on: Nov 26, 2019

Published on: Apr 16, 2020

Published by: Society of Nematologists, Inc.

In partnership with: Paradigm Publishing Services

Publication frequency: 1 issue per year

Keywords:

transcriptome,

next generation sequencing,

differentially expressed genes,

genomics

Related subjects:

Life sciences,

Life sciences, other

© 2020 U-Hwa Nam, Jong-Oh Kim, Jeong-Ho Kim, published by Society of Nematologists, Inc.
This work is licensed under the Creative Commons Attribution 4.0 License.

Previous article Volume 52 (2020): Issue 1 (January 2020)Next article

De novo transcriptome sequencing and analysis of Anisakis pegreffii (Nematoda: Anisakidae) third-stage and fourth stage larvae

Figures & Tables

Figure 1:

Figure 2:

Top 20 most differentially expressed genes in A_ pegreffi L3_

Gene expression overview of A_ pegreffii L3 and L4 transcriptomes_

Preprocessing statistics of Anisakis transcriptome_

Top 20 most abundant unigenes in A_ pegreffii L4_

Top 20 most abundant unigenes in A_ pegreffii L3_

Primer information for real-time PCR analysis_

Top 20 most differentially expressed genes in A_ pegreffi L4_

Paradigm

My account