KaWai_RNASeq_removeZero.md

RNA-Seq data from Ka-Wai Ma

• 1. pWER agar plate
  • 1.1 Alignment
  • 1.2 DEGs
  • 1.3 Gene-set
• 2. Col0 agar plate
  • 2.1 Alignment
  • 2.2 DEGs
  • 2.3 Gene-set
• 3. Col0 flow pot
  • 3.1 Alignment
  • 3.2 DEGs
  • 3.3 Gene-set
• 4. Merge
  • 4.1 WER Col0 agar plate
  • 4.2 Col0 agar flowpot
  • 4.3 Col0 agar flowpot iron
  • 4.4 Col0 agar flowpot flg22
• References

1. pWER agar plate

1.1 Alignment

sample	rawfq	trimfq	H_ath	K_ath
Mock_1	16978228	16881903	0.977	0.962
Mock_2	18851317	18723047	0.979	0.966
Mock_3	18313224	18218407	0.98	0.966
Flg22_1	17512255	17422675	0.971	0.954
Flg22_2	18047704	17950283	0.975	0.959
Flg22_3	17922052	17826125	0.974	0.957
SynCom33_Flg22_1	17785611	17687693	0.974	0.955
SynCom33_Flg22_2	16929833	16832003	0.975	0.96
SynCom33_Flg22_3	20093432	19984770	0.974	0.956
SynCom35_Flg22_1	17361283	17267286	0.965	0.952
SynCom35_Flg22_2	16914038	16825207	0.979	0.966
SynCom35_Flg22_3	21203518	21099083	0.972	0.957

1.2 DEGs

Remove 0|0|0 or 0|0|x, then 29163 transcripts were kept.

• PCA plot

PCA plot of raw data

PCA plot of SVA corrected data (3 hidden variables)

• Cluster

• DEGs groups

cond <- list(c('Mock_Flg22', 'Mock'),
             c('SynCom33_Flg22', 'Mock'),
             c('SynCom35_Flg22', 'Mock'),
             c('SynCom33_Flg22', 'Mock_Flg22'),
             c('SynCom35_Flg22', 'Mock_Flg22'),
             c('SynCom33_Flg22', 'SynCom35_Flg22'))

DEGs threshold: |log2FC| > log2(1.5) and adj-pvalue < 0.05

DEGs table

• Heatmap

1.3 Gene-set

GO analysis

• dotplot

• Network (top 5 significant GO terms)

• Optimized network (top 5 significant GO terms)

• Expand GO terms (top 10 significant GO terms)

• Optimized network (top 10 significant GO terms)

2. Col0 agar plate

2.1 Alignment

sample	rawfq_R1	rawfq_R2	trimfq	H_ath	K_ath	Hvirus_ath	Kvirus_ath
HKSynCom33_1	13172195	13172195	12783484	0.465	0.458	0.975	0.981
HKSynCom33_2	10767044	10767044	10287182	0.652	0.643	0.976	0.975
HKSynCom33_3	10878033	10878033	10560115	0.58	0.572	0.974	0.976
HKSynCom33_4	8651910	8651910	8454418	0.83	0.816	0.98	0.969
HKSynCom33_Flg22_1	6075012	6075012	5922387	0.887	0.871	0.978	0.964
HKSynCom33_Flg22_2	11499848	11499848	11142660	0.552	0.542	0.975	0.975
HKSynCom33_Flg22_3	11608298	11608298	11242512	0.718	0.71	0.976	0.975
HKSynCom33_Flg22_4	8763571	8763571	8519488	0.624	0.615	0.979	0.976
HKSynCom35_1	10084160	10084160	9826277	0.809	0.796	0.976	0.966
HKSynCom35_2	6297490	6297490	6170945	0.912	0.9	0.98	0.969
HKSynCom35_3	9915057	9915057	9603304	0.727	0.722	0.977	0.977
HKSynCom35_4	12587610	12587610	12091544	0.563	0.55	0.971	0.973
HKSynCom35_Flg22_1	6873637	6873637	6588694	0.977	0.963	0.977	0.963
HKSynCom35_Flg22_2	6461144	6461144	6307990	0.814	0.803	0.979	0.971
HKSynCom35_Flg22_3	8719346	8719346	8516934	0.6	0.595	0.977	0.98
HKSynCom35_Flg22_4	12973482	12973482	12618526	0.538	0.526	0.977	0.975
Mock_1	13036385	13036385	12732898	0.559	0.549	0.979	0.978
Mock_2	11495909	11495909	11144820	0.752	0.745	0.976	0.973
Mock_3	14230460	14230460	13715287	0.51	0.503	0.974	0.978
Mock_4	12354390	12354390	11513757	0.592	0.584	0.922	0.922
Mock_Flg22_1	11018794	11018794	10700223	0.703	0.693	0.978	0.973
Mock_Flg22_2	11452528	11452528	11123514	0.644	0.637	0.975	0.975
Mock_Flg22_3	13355598	13355598	12868862	0.579	0.571	0.974	0.977
Mock_Flg22_4	10720255	10720255	10352975	0.8	0.786	0.978	0.968
SynCom33_1	10665251	10665251	10238121	0.729	0.718	0.976	0.971
SynCom33_2	13777849	13777849	13220235	0.502	0.497	0.975	0.981
SynCom33_3	12414664	12414664	12026972	0.668	0.664	0.977	0.978
SynCom33_4	12574124	12574124	12030929	0.634	0.623	0.975	0.972
SynCom33_Flg22_1	11354523	11354523	11037970	0.67	0.661	0.976	0.974
SynCom33_Flg22_2	11098796	11098796	10723034	0.64	0.629	0.977	0.973
SynCom33_Flg22_3	12743113	12743113	12297414	0.536	0.531	0.878	0.881
SynCom33_Flg22_4	9369409	9369409	9138305	0.671	0.66	0.977	0.972
SynCom35_1	11683102	11683102	11384694	0.636	0.623	0.978	0.974
SynCom35_2	10254853	10254853	9911636	0.825	0.814	0.977	0.97
SynCom35_3	8767980	8767980	8025669	0.802	0.797	0.972	0.972
SynCom35_4	12844974	12844974	11942099	0.617	0.606	0.973	0.971
SynCom35_Flg22_1	11048570	11048570	10706105	0.614	0.603	0.977	0.974
SynCom35_Flg22_2	10988696	10988696	10515421	0.635	0.627	0.975	0.975
SynCom35_Flg22_3	8719287	8719287	8476158	0.707	0.698	0.977	0.974
SynCom35_Flg22_4	11436243	11436243	11120876	0.689	0.672	0.979	0.967

2.2 DEGs

Remove 0|0|0|0 or 0|0|0|x, then 27592 transcripts were kept.

DEGs table

• PCA plot

PCA plot of raw data

PCA plot of SVA corrected data (3 hidden variables)

• Cluster

• DEGs groups

## flg22 treatment effect
cond1 <- list(c('Mock_Flg22', 'Mock'),
             c('HKSynCom33_Flg22', 'HKSynCom33'),
             c('HKSynCom35_Flg22', 'HKSynCom35'),
             c('SynCom33_Flg22', 'SynCom33'),
             c('SynCom35_Flg22', 'SynCom35'))

## bacteria effect
cond2 <- list(c('HKSynCom33', 'Mock'),
              c('HKSynCom35', 'Mock'),
              c('SynCom33', 'Mock'),
              c('SynCom35', 'Mock'),
              c('SynCom33', 'SynCom35'),
              c('HKSynCom33', 'HKSynCom35'))

## bacteria x flg22 effect
cond3 <- list(c('HKSynCom33_Flg22', 'Mock'),
              c('HKSynCom35_Flg22', 'Mock'),
              c('SynCom33_Flg22', 'Mock'),
              c('SynCom35_Flg22', 'Mock'),
              c('SynCom33_Flg22', 'SynCom35_Flg22'),
              c('HKSynCom33_Flg22', 'HKSynCom35_Flg22'),
              c('HKSynCom33_Flg22', 'Mock_Flg22'),
              c('HKSynCom35_Flg22', 'Mock_Flg22'),
              c('SynCom33_Flg22', 'Mock_Flg22'),
              c('SynCom35_Flg22', 'Mock_Flg22'))

## heat kill effect
cond4 <- list(c('SynCom33', 'HKSynCom33'),
              c('SynCom35', 'HKSynCom35'))

DEGs threshold: |log2FC| > log2(1.5) and adj-pvalue < 0.05

• Heatmap

2.3 Gene-set

GO analysis

• dotplot

• Network (top 5 significant GO terms)

• Optimized network (top 5 significant GO terms)

• Expand GO terms (top 10 significant GO terms)

• Optimized network (top 10 significant GO terms)

3. Col0 flow pot

3.1 Alignment

sample	rawfq_R1	rawfq_R2	trimfq	H_ath	K_ath	Hvirus_ath	Kvirus_ath
Mock_1	8146907	8146907	7877992	0.743	0.733	0.967	0.963
Mock_2	18083448	18083448	17391986	0.245	0.241	0.96	0.98
Mock_3	12919098	12919098	12363313	0.468	0.465	0.962	0.974
SynCom33_1	13209898	13209898	12648097	0.406	0.401	0.959	0.97
SynCom33_2	15867598	15867598	15210307	0.277	0.274	0.968	0.986
SynCom33_3	12568370	12568370	12105344	0.437	0.435	0.968	0.98
SynCom35_1	10898141	10898141	10483412	0.652	0.649	0.969	0.975
SynCom35_2	16818587	16818587	16215582	0.341	0.338	0.965	0.98
SynCom35_3	11391744	11391744	10974092	0.57	0.566	0.967	0.973

3.2 DEGs

Remove 0|0|0 or 0|0|x, then 27105 transcripts were kept.

DEGs table

• PCA plot

PCA plot of raw data

PCA plot of SVA corrected data (3 hidden variables)

• Cluster

• DEGs groups

cond <- list(c('SynCom33', 'Mock'),
             c('SynCom35', 'Mock'),
             c('SynCom33', 'SynCom35'))

• Heatmap

3.3 Gene-set

GO analysis

• dotplot

• Network (top 5 significant GO terms)

• Optimized network (top 5 significant GO terms)

• Expand GO terms (top 10 significant GO terms)

• Optimized network (top 10 significant GO terms)

4. Merge

4.1 WER Col0 agar plate

4.2 Col0 agar flowpot

4.3 Col0 agar flowpot iron

4.4 Col0 agar flowpot flg22

References

• Garrido-Oter R, Nakano RT, Dombrowski N, Ma KW; AgBiome Team, McHardy AC, Schulze-Lefert P. Modular Traits of the Rhizobiales Root Microbiota and Their Evolutionary Relationship with Symbiotic Rhizobia Cell Host Microbe. 2018;24(1):155-167.e5.