Immunoglobulin Repertoire Sequencing Analysis Report

After quality trimming, each Read1 and Read2 sequence pair was merged based on overlapping sequences. Merged reads with length less than 200bp were removed from subsequent analysis. The statistics of each processing step are summarized in Table 3.1. Length distribution of the merged contigs is shown in Figure 3.2.

Table 3.1 Raw sequencing data quality statistics

Sample	Raw Count	% of Raw(>Q20)	% of Raw(>Q30)	Clean Count	% of Clean(>Q20)	% of Clean(>Q30)	% of Trimmed	Merged Count	% of Merged	Mapped Count	% of Mapped
Sample	Raw_count	%_of_rawQ20	%_of_rawQ30	Clean_count	%_of_cleanQ20	%_of_cleanQ30	%_of_trimmed	Merged_count	%_of_merged	Mapped_count	%_of_mapped
ASC-S-vH	1e+06	83.31	70.39	420350	99.08	94.94	42.03	300554	30.06	172313	57.33
ASC-S-vL	1e+06	85.27	73.77	447107	99.05	94.96	44.71	333677	33.37	119172	35.71
PBMC-0-S-vH	1e+06	83.78	71.34	448438	99.09	94.97	44.84	283774	28.38	173686	61.21
PBMC-0-S-vL	1e+06	85.08	73.35	431477	99.03	94.91	43.15	319008	31.9	114945	36.03
PBMC-7-S-vH	1e+06	83.71	71	439184	99.09	94.97	43.92	297230	29.72	171697	57.77
PBMC-7-S-vL	1e+06	85.15	73.38	427074	99.03	94.93	42.71	320174	32.02	116052	36.25

 

4. Immunoglobulin Sequence Analysis

 

4.1 Sequence Alignment

The assembled reads were blasted against IMGT database to identify the best germline V(D)J gene matches [5]. The alignment result for each individual sequences is as in Table 4.1. The complete output for all the samples are in the ‘Report\Clonal_tables’ directory and can be accessed using the link below:

  Clonal tables

 

4.2 CDR Abundancy Analysis

To assess sequence abundancy, reads with the same CDR sequences were collapsed together and the counts of each unique sequences were calculated. The aggregation was performed on three levels – sequences were first collapsed based on nucleotide sequences of CDR1,2,3 (Tables 4.2.1 and 4.2.2), then further collapsed based on CDR1,2,3 amino acid sequences (Tables 4.2.3 and 4.2.4), and then further collapsed based on CDR3 amino acid sequence alone (Tables 4.2.5 and 4.2.6). The complete CDR1,2,3 enrichment analysis for all the samples are in “CDR_all_enrichment” directory, the CDR amino acid sequence enrichment analysis for all the samples are in “CDR_aa_enrichment” directory, and the CDR3 enrichment analysis for all the samples are in “CDR3_aa_enrichment” directory in the the Report/Enrichment_tables/ folder or can be accessed using the link below:

  CDR_all_enrichment
  CDR_aa_enrichment
  CDR3_aa_enrichment

 

4.3 CDR3 Length Distribution

CDR3 amino acid sequences were further extracted and the length distribution was plotted as in Figure 4.3.

 

4.4 Top V(D)J Analysis

V(D)J usage of sequences were analyzed. Sequences were collapsed on V(D)J combination. The proportions of the top ten combinations in the heavy and light chain populations are shown in Figure 4.4.1 and Figure 4.4.2, respectively.

 

4.5 V Gene Usage Analysis

To assess germline gene usage, V gene annotation information was extracted for all the sequences and the total count of each germline gene was calculated and summarized in Tables 4.5.1 and 4.5.2.

Table 4.5.1 Heavy chain V gene usage

Top_V_Gene	ASC-S-vH	PBMC-0-S-vH	PBMC-7-S-vH
IGHV1	26385	33920	28208
IGHV10	1418	1868	1583
IGHV10S3	16	10	7
IGHV10S4	1	NA	1
IGHV12	3858	2548	3551
IGHV13	31	4	20
IGHV14	919	1738	1189
IGHV14S4	2	NA	NA
IGHV15	21	NA	NA
IGHV16	612	386	406
IGHV1S12	577	258	322
IGHV1S126	647	523	317
IGHV1S127	510	436	250
IGHV1S130	3	4	NA
IGHV1S132	10	15	4
IGHV1S134	38	15	68
IGHV1S136	4	13	28
IGHV1S137	1	1	9
IGHV1S14	2	NA	1
IGHV1S15	51	18	109
IGHV1S17	1	1	15
IGHV1S18	7	258	141
IGHV1S19	4	12	103
IGHV1S22	20	19	2
IGHV1S26	697	270	322
IGHV1S28	21	21	6
IGHV1S29	14	26	6
IGHV1S34	4	14	12
IGHV1S45	190	25	120
IGHV1S46	408	1032	600
IGHV1S5	2	7	2
IGHV1S50	31	83	58
IGHV1S55	7	2	6
IGHV1S56	178	146	635
IGHV1S61	48	53	41
IGHV1S81	111	55	24
IGHV2	42	21	21
IGHV3	61675	40187	56460
IGHV3S1	8800	21984	15880
IGHV3S7	16	2	12
IGHV4	22	1	14
IGHV5	35995	35220	36499
IGHV5S12	31	77	103
IGHV5S21	266	166	113
IGHV5S24	18	1	11
IGHV5S4	59	9	44
IGHV5S9	1	2	8
IGHV6	455	333	428
IGHV7	1481	1333	1406
IGHV8	26539	30520	22482
IGHV8S2	28	9	6
IGHV9	36	37	44
IGHV1S16	NA	1	NA
IGHV1S47	NA	1	NA
IGHV2S3	NA	1	NA



Table 4.5.2 Light chain V gene usage

Top_V_Gene	ASC-S-vL	PBMC-0-S-vL	PBMC-7-S-vL
IGKV1	7420	4924	5979
IGKV10	506	309	351
IGKV11	49516	36390	41839
IGKV12	4256	7743	5958
IGKV13	115	53	85
IGKV14	327	128	245
IGKV15	1261	1100	1443
IGKV16	3775	2143	1970
IGKV17	2	3	2
IGKV18	31877	42729	38078
IGKV19	127	43	24
IGKV2	1159	1714	1566
IGKV3	62	37	57
IGKV4	100	18	32
IGKV5	10033	8702	8996
IGKV6	483	744	597
IGKV8	8066	8131	8794
IGKV9	85	27	34
IGLV1	NA	2	NA



The distribution of V gene usage is also illustrated in bar graphs (Figure 4.5.1 and Figure 4.5.2).

 

4.6 Clustering Analysis

To analyze CDR3 amino acid usage frequency, CDR3 sequences were clustered according to similarity (threshold: 0.8). The summary of top 20 clusters of each sample is as in Table 4.6. The complete results for all the samples are in the 'Report\Clustering_summary_tables' folder and can be accessed using the link below:

  Clustering tables

The logo of the most representative sequence of each cluster [6] is in the 'Report\Clustering_weblogo' folder and can be viewed using the links below:

  Weblogo

 

4.7 Diversity Analysis

The clonal abundance distribution was calculated with confidence intervals derived via bootstrapping. The clonal diversity of the repertoire was accessed using diversity index [7]. Abundance curves are in figure 4.7.1 and diversity curves are in Figure 4.7.2.

 

5. References

[1] Murphy K. Janeway's Immunobiology. New York: Garland Science (2012).

[2] Lefranc, MP. et al., The international ImMunoGeneTics database. Nucleic Acids Res. (1999) 27 (1): 209-212.

[3] Bolger, AM. et al., Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. (2014) 30(15): 2114–2120.

[4] Andrews S. et al., FastQC: a quality control tool for high throughput sequence data. (2010) Available online at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc.

[5] Ye J. et al., IgBLAST: an immunoglobulin variable domain sequence analysis tool. Nucleic Acids Res. (2013) 41: W34–W40.

[6] Crooks GE. et al., WebLogo: a sequence logo generator. Genome Res. (2004) 14(6):1188-90.

[7] Hill, M. et al., Diversity and evenness: a unifying notation and its consequences. Ecology (1973) 54:427-432.