title	author	output	date
Tipton_2015_analysis_part1_v01-01	William Guesdon	html_document code_folding toc df_print hide true kable	Compiled: `r format(Sys.Date(), "%B %d, %Y")`

```{r setup, include=FALSE} knitr::opts_chunk$set(echo = TRUE) ``` ```{r warning=FALSE, message=FALSE} ################ # Load libraries ################ library(tidyverse) library(alakazam) library(shazam) library(cowplot) library(rstatix) library(cowplot) library(ggpubr) ``` ```{r echo=FALSE, warning=FALSE, message=FALSE, include=FALSE} ##################### #Set up ggplot theme #################### tmp_theme Tipton_2015_FLU_Bulk_FLU_subject_1_iglblastn_Bulk.fasta Tipton_2015_FLU_Bulk_FLU_subject_1_iglblastn_Bulk.fasta.gz Tipton_2015_FLU_Bulk_FLU_subject_1_iglblastn_Bulk.json.gz Tipton_2015_FLU_Bulk_FLU_subject_1_iglblastn_Bulk.txz Tipton_2015_FLU_Bulk_FLU_subject_2_iglblastn_Bulk.fasta Tipton_2015_FLU_Bulk_FLU_subject_2_iglblastn_Bulk.fasta.gz Tipton_2015_FLU_Bulk_FLU_subject_2_iglblastn_Bulk.json.gz Tipton_2015_FLU_Bulk_FLU_subject_2_iglblastn_Bulk.txz Tipton_2015_FLU_Bulk_FLU_subject_3_iglblastn_Bulk.fasta Tipton_2015_FLU_Bulk_FLU_subject_3_iglblastn_Bulk.fasta.gz Tipton_2015_FLU_Bulk_FLU_subject_3_iglblastn_Bulk.json.gz Tipton_2015_FLU_Bulk_FLU_subject_3_iglblastn_Bulk.txz Tipton_2015_FLU_Bulk_FLU_subject_4_iglblastn_Bulk.fasta Tipton_2015_FLU_Bulk_FLU_subject_4_iglblastn_Bulk.fasta.gz Tipton_2015_FLU_Bulk_FLU_subject_4_iglblastn_Bulk.json.gz Tipton_2015_FLU_Bulk_FLU_subject_4_iglblastn_Bulk.txz Tipton_2015_TET_Bulk_TET_subject_1_iglblastn_Bulk.fasta Tipton_2015_TET_Bulk_TET_subject_1_iglblastn_Bulk.fasta.gz Tipton_2015_TET_Bulk_TET_subject_1_iglblastn_Bulk.json.gz Tipton_2015_TET_Bulk_TET_subject_1_iglblastn_Bulk.txz Tipton_2015_TET_Bulk_TET_subject_2_iglblastn_Bulk.fasta Tipton_2015_TET_Bulk_TET_subject_2_iglblastn_Bulk.fasta.gz Tipton_2015_TET_Bulk_TET_subject_2_iglblastn_Bulk.json.gz Tipton_2015_TET_Bulk_TET_subject_2_iglblastn_Bulk.txz Tipton_2015_TET_Bulk_TET_subject_3_iglblastn_Bulk.fasta Tipton_2015_TET_Bulk_TET_subject_3_iglblastn_Bulk.fasta.gz Tipton_2015_TET_Bulk_TET_subject_3_iglblastn_Bulk.json.gz Tipton_2015_TET_Bulk_TET_subject_3_iglblastn_Bulk.txz Tipton_2015_TET_Bulk_TET_subject_4_iglblastn_Bulk.fasta Tipton_2015_TET_Bulk_TET_subject_4_iglblastn_Bulk.fasta.gz Tipton_2015_TET_Bulk_TET_subject_4_iglblastn_Bulk.json.gz Tipton_2015_TET_Bulk_TET_subject_4_iglblastn_Bulk.txz cd .. cd cd SLE/ ls > Tipton_2015_SLE_Bulk_SLE_subject_1_iglblastn_Bulk.fasta Tipton_2015_SLE_Bulk_SLE_subject_1_iglblastn_Bulk.fasta.gz Tipton_2015_SLE_Bulk_SLE_subject_1_iglblastn_Bulk.json.gz Tipton_2015_SLE_Bulk_SLE_subject_1_iglblastn_Bulk.txz Tipton_2015_SLE_Bulk_SLE_subject_2_iglblastn_Bulk.fasta Tipton_2015_SLE_Bulk_SLE_subject_2_iglblastn_Bulk.fasta.gz Tipton_2015_SLE_Bulk_SLE_subject_2_iglblastn_Bulk.json.gz Tipton_2015_SLE_Bulk_SLE_subject_2_iglblastn_Bulk.txz Tipton_2015_SLE_Bulk_SLE_subject_3_iglblastn_Bulk.fasta Tipton_2015_SLE_Bulk_SLE_subject_3_iglblastn_Bulk.fasta.gz Tipton_2015_SLE_Bulk_SLE_subject_3_iglblastn_Bulk.json.gz Tipton_2015_SLE_Bulk_SLE_subject_3_iglblastn_Bulk.txz Tipton_2015_SLE_Bulk_SLE_subject_4_iglblastn_Bulk.fasta Tipton_2015_SLE_Bulk_SLE_subject_4_iglblastn_Bulk.fasta.gz Tipton_2015_SLE_Bulk_SLE_subject_4_iglblastn_Bulk.json.gz Tipton_2015_SLE_Bulk_SLE_subject_4_iglblastn_Bulk.txz Tipton_2015_SLE_Bulk_SLE_subject_5_iglblastn_Bulk.fasta Tipton_2015_SLE_Bulk_SLE_subject_5_iglblastn_Bulk.fasta.gz Tipton_2015_SLE_Bulk_SLE_subject_5_iglblastn_Bulk.json.gz Tipton_2015_SLE_Bulk_SLE_subject_5_iglblastn_Bulk.txz Tipton_2015_SLE_Bulk_SLE_subject_6_iglblastn_Bulk.fasta Tipton_2015_SLE_Bulk_SLE_subject_6_iglblastn_Bulk.fasta.gz Tipton_2015_SLE_Bulk_SLE_subject_6_iglblastn_Bulk.json.gz Tipton_2015_SLE_Bulk_SLE_subject_6_iglblastn_Bulk.txz Tipton_2015_SLE_Bulk_SLE_subject_7_iglblastn_Bulk.fasta Tipton_2015_SLE_Bulk_SLE_subject_7_iglblastn_Bulk.fasta.gz Tipton_2015_SLE_Bulk_SLE_subject_7_iglblastn_Bulk_fasta.txz Tipton_2015_SLE_Bulk_SLE_subject_7_iglblastn_Bulk.json.gz Tipton_2015_SLE_Bulk_SLE_subject_8_iglblastn_Bulk.fasta Tipton_2015_SLE_Bulk_SLE_subject_8_iglblastn_Bulk.fasta.gz Tipton_2015_SLE_Bulk_SLE_subject_8_iglblastn_Bulk.json.gz Tipton_2015_SLE_Bulk_SLE_subject_8_iglblastn_Bulk.txz ###################### # 2 Parse IMGT output ###################### cd data/HC/ MakeDb.py imgt -i Tipton_2015_TET_Bulk_TET_subject_1_iglblastn_Bulk.txz -s Tipton_2015_TET_Bulk_TET_subject_1_iglblastn_Bulk.fasta --regions --scores > START> MakeDb ALIGNER> IMGT ALIGNER_FILE> Tipton_2015_TET_Bulk_TET_subject_1_iglblastn_Bulk.txz SEQ_FILE> Tipton_2015_TET_Bulk_TET_subject_1_iglblastn_Bulk.fasta ASIS_ID> False PARTIAL> False SCORES> True REGIONS> True JUNCTION> False PROGRESS> 18:43:29 |Done | 0.0 min PROGRESS> 18:43:33 |####################| 100% (18,576) 0.1 min OUTPUT> Tipton_2015_TET_Bulk_TET_subject_1_iglblastn_Bulk_db-pass.tab PASS> 1039 FAIL> 17537 END> MakeDb MakeDb.py imgt -i Tipton_2015_TET_Bulk_TET_subject_2_iglblastn_Bulk.txz -s Tipton_2015_TET_Bulk_TET_subject_2_iglblastn_Bulk.fasta --regions --scores > START> MakeDb ALIGNER> IMGT ALIGNER_FILE> Tipton_2015_TET_Bulk_TET_subject_2_iglblastn_Bulk.txz SEQ_FILE> Tipton_2015_TET_Bulk_TET_subject_2_iglblastn_Bulk.fasta ASIS_ID> False PARTIAL> False SCORES> True REGIONS> True JUNCTION> False PROGRESS> 18:44:17 |Done | 0.0 min PROGRESS> 18:44:20 |####################| 100% (10,196) 0.0 min OUTPUT> Tipton_2015_TET_Bulk_TET_subject_2_iglblastn_Bulk_db-pass.tab PASS> 668 FAIL> 9528 END> MakeDb MakeDb.py imgt -i Tipton_2015_TET_Bulk_TET_subject_3_iglblastn_Bulk.txz -s Tipton_2015_TET_Bulk_TET_subject_3_iglblastn_Bulk.fasta --regions --scores > START> MakeDb ALIGNER> IMGT ALIGNER_FILE> Tipton_2015_TET_Bulk_TET_subject_3_iglblastn_Bulk.txz SEQ_FILE> Tipton_2015_TET_Bulk_TET_subject_3_iglblastn_Bulk.fasta ASIS_ID> False PARTIAL> False SCORES> True REGIONS> True JUNCTION> False PROGRESS> 18:44:59 |Done | 0.0 min PROGRESS> 18:45:00 |####################| 100% (3,532) 0.0 min OUTPUT> Tipton_2015_TET_Bulk_TET_subject_3_iglblastn_Bulk_db-pass.tab PASS> 1585 FAIL> 1947 END> MakeDb MakeDb.py imgt -i Tipton_2015_TET_Bulk_TET_subject_4_iglblastn_Bulk.txz -s Tipton_2015_TET_Bulk_TET_subject_4_iglblastn_Bulk.fasta --regions --scores > START> MakeDb ALIGNER> IMGT ALIGNER_FILE> Tipton_2015_TET_Bulk_TET_subject_4_iglblastn_Bulk.txz SEQ_FILE> Tipton_2015_TET_Bulk_TET_subject_4_iglblastn_Bulk.fasta ASIS_ID> False PARTIAL> False SCORES> True REGIONS> True JUNCTION> False PROGRESS> 18:45:53 |Done | 0.0 min PROGRESS> 18:45:59 |####################| 100% (24,445) 0.1 min OUTPUT> Tipton_2015_TET_Bulk_TET_subject_4_iglblastn_Bulk_db-pass.tab PASS> 1810 FAIL> 22635 END> MakeDb cd .. cd cd SLE/ MakeDb.py imgt -i Tipton_2015_SLE_Bulk_SLE_subject_1_iglblastn_Bulk.txz -s Tipton_2015_SLE_Bulk_SLE_subject_1_iglblastn_Bulk.fasta --regions --scores > START> MakeDb ALIGNER> IMGT ALIGNER_FILE> Tipton_2015_SLE_Bulk_SLE_subject_1_iglblastn_Bulk.txz SEQ_FILE> Tipton_2015_SLE_Bulk_SLE_subject_1_iglblastn_Bulk.fasta ASIS_ID> False PARTIAL> False SCORES> True REGIONS> True JUNCTION> False PROGRESS> 18:48:01 |Done | 0.0 min PROGRESS> 18:48:06 |####################| 100% (21,893) 0.1 min OUTPUT> Tipton_2015_SLE_Bulk_SLE_subject_1_iglblastn_Bulk_db-pass.tab PASS> 912 FAIL> 20981 END> MakeDb MakeDb.py imgt -i Tipton_2015_SLE_Bulk_SLE_subject_2_iglblastn_Bulk.txz -s Tipton_2015_SLE_Bulk_SLE_subject_2_iglblastn_Bulk.fasta --regions --scores > START> MakeDb ALIGNER> IMGT ALIGNER_FILE> Tipton_2015_SLE_Bulk_SLE_subject_2_iglblastn_Bulk.txz SEQ_FILE> Tipton_2015_SLE_Bulk_SLE_subject_2_iglblastn_Bulk.fasta ASIS_ID> False PARTIAL> False SCORES> True REGIONS> True JUNCTION> False PROGRESS> 18:50:56 |Done | 0.0 min PROGRESS> 18:51:00 |####################| 100% (15,164) 0.1 min OUTPUT> Tipton_2015_SLE_Bulk_SLE_subject_2_iglblastn_Bulk_db-pass.tab PASS> 1012 FAIL> 14152 END> MakeDb MakeDb.py imgt -i Tipton_2015_SLE_Bulk_SLE_subject_3_iglblastn_Bulk.txz -s Tipton_2015_SLE_Bulk_SLE_subject_3_iglblastn_Bulk.fasta --regions --scores > START> MakeDb ALIGNER> IMGT ALIGNER_FILE> Tipton_2015_SLE_Bulk_SLE_subject_3_iglblastn_Bulk.txz SEQ_FILE> Tipton_2015_SLE_Bulk_SLE_subject_3_iglblastn_Bulk.fasta ASIS_ID> False PARTIAL> False SCORES> True REGIONS> True JUNCTION> False PROGRESS> 18:51:39 |Done | 0.2 min PROGRESS> 18:52:19 |####################| 100% (172,694) 0.7 min OUTPUT> Tipton_2015_SLE_Bulk_SLE_subject_3_iglblastn_Bulk_db-pass.tab PASS> 8162 FAIL> 164532 END> MakeDb MakeDb.py imgt -i Tipton_2015_SLE_Bulk_SLE_subject_4_iglblastn_Bulk.txz -s Tipton_2015_SLE_Bulk_SLE_subject_4_iglblastn_Bulk.fasta --regions --scores > START> MakeDb ALIGNER> IMGT ALIGNER_FILE> Tipton_2015_SLE_Bulk_SLE_subject_4_iglblastn_Bulk.txz SEQ_FILE> Tipton_2015_SLE_Bulk_SLE_subject_4_iglblastn_Bulk.fasta ASIS_ID> False PARTIAL> False SCORES> True REGIONS> True JUNCTION> False PROGRESS> 18:53:04 |Done | 0.0 min PROGRESS> 18:53:14 |####################| 100% (39,846) 0.2 min OUTPUT> Tipton_2015_SLE_Bulk_SLE_subject_4_iglblastn_Bulk_db-pass.tab PASS> 959 FAIL> 38887 END> MakeDb MakeDb.py imgt -i Tipton_2015_SLE_Bulk_SLE_subject_5_iglblastn_Bulk.txz -s Tipton_2015_SLE_Bulk_SLE_subject_5_iglblastn_Bulk.fasta --regions --scores > START> MakeDb ALIGNER> IMGT ALIGNER_FILE> Tipton_2015_SLE_Bulk_SLE_subject_5_iglblastn_Bulk.txz SEQ_FILE> Tipton_2015_SLE_Bulk_SLE_subject_5_iglblastn_Bulk.fasta ASIS_ID> False PARTIAL> False SCORES> True REGIONS> True JUNCTION> False PROGRESS> 18:53:42 |Done | 0.0 min PROGRESS> 18:53:48 |####################| 100% (25,157) 0.1 min OUTPUT> Tipton_2015_SLE_Bulk_SLE_subject_5_iglblastn_Bulk_db-pass.tab PASS> 1919 FAIL> 23238 END> MakeDb MakeDb.py imgt -i Tipton_2015_SLE_Bulk_SLE_subject_6_iglblastn_Bulk.txz -s Tipton_2015_SLE_Bulk_SLE_subject_6_iglblastn_Bulk.fasta --regions --scores > START> MakeDb ALIGNER> IMGT ALIGNER_FILE> Tipton_2015_SLE_Bulk_SLE_subject_6_iglblastn_Bulk.txz SEQ_FILE> Tipton_2015_SLE_Bulk_SLE_subject_6_iglblastn_Bulk.fasta ASIS_ID> False PARTIAL> False SCORES> True REGIONS> True JUNCTION> False PROGRESS> 18:54:24 |Done | 0.0 min PROGRESS> 18:54:26 |####################| 100% (8,558) 0.0 min OUTPUT> Tipton_2015_SLE_Bulk_SLE_subject_6_iglblastn_Bulk_db-pass.tab PASS> 2997 FAIL> 5561 END> MakeDb ``` **A majority of the sequecnes failed. This could be caused by a difference in the pipeline check the in the paper method what sequencing methd was used.** Library preparation: NGS data is deposited at the NCBI sequence read archive (SRA) study accession, SRP057017. Total cellular RNA was isolated from the number of cells outlined in Table 1 using the RNeasy Micro kit by following the manufacturer's protocol (Qiagen). Approximately 2 ng of RNA was subjected to reverse transcription using the iScript cDNA synthesis kit (BioRad). Aliquots of the resulting single-stranded cDNA products were mixed with 50 nM of VH1-VH7 FR1 specific primers and 250 nM Cα, Cμ, and Cγ specific primers preceded by the respective Illumina nextera sequencing tag (oligonucleotide sequences listed below) in a 25 μl PCR reaction (using 4 αl template cDNA) using Invitrogen's High Fidelity Platinum PCR Supermix (Invitrogen). # Lod each file, edit table and fuse * Load the files * Add the variables * Donor * Health Status HC or SLE * Isotype * Fuse all the table in one master table * Run the rest of Immcantation pipeline on the master table ```{r} HC1 % dplyr::mutate( Donor = counter, Status = "HC" ) counter % dplyr::mutate( Donor = counter, Status = "SLE" ) counter % filter(FUNCTIONAL == "TRUE") HC % filter(FUNCTIONAL == "TRUE") all_samples all_HC.tab all_samples.tab all_SLE.tab shazam-threshold -d all_HC.tab > THRESHOLD_AVG> 0.0674195 shazam-threshold -d all_SLE.tab > THRESHOLD_AVG> 0.0674195 shazam-threshold -d all_samples.tab > THRESHOLD_AVG> 0.0592632 ``` # Why is the threshold EXACTLY the same for SLE and HC? Check that the 2 df are different ```{r} glimpse(HC) #View(HC) ``` ```{r} glimpse(SLE) #View(SLE) ``` ```{r} glimpse(all_samples) #View(all_samples) ``` Did not used parse select to remove non functional sequence but instead directly use dplyr. After verification it appears that the dataset are clearly differents ```{bash eval=FALSE} #################### # 5 Clonal threshold #################### changeo-clone -d all_samples.tab -x 0.0592632 > START 1: ParseDb select 14:01 01/22/20 2: DefineClones 14:01 01/22/20 3: CreateGermlines 14:05 01/22/20 4: ParseLog 14:06 01/22/20 5: Compressing files 14:06 01/22/20 DONE ```