HAL Id: hal-01132460
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01132460
Submitted on 26 Jun 2015
HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from
L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de
Somatic mutations of cell-free circulating DNA detected by Next Generation Sequencing reflect the genetic
changes in both Germinal Center B-Cell like and Activated B-Cell like Diffuse Large B-Cell Lymphoma
tumors at the time of diagnosis
Elodie Bohers, Pierre Julien Viailly, Sydney Dubois, Philippe Bertrand, Catherine Maingonnat, Sylvain Mareschal, Philippe Ruminy, Jean-Michel
Picquenot, Christian Bastard, Fabienne Desmots, et al.
To cite this version:
Elodie Bohers, Pierre Julien Viailly, Sydney Dubois, Philippe Bertrand, Catherine Maingonnat, et al..
Somatic mutations of cell-free circulating DNA detected by Next Generation Sequencing reflect the genetic changes in both Germinal Center B-Cell like and Activated B-Cell like Diffuse Large B-Cell Lymphoma tumors at the time of diagnosis. Haematologica, Ferrata Storti Foundation, 2015, 100 (7), pp.e280-e284. �10.3324/haematol.2015.123612�. �hal-01132460�
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Supplementary file 1. Lymphopanel and PGM data analysis
A. Lymphopanel set used for NGS experiments
Gene Transcript Reference Hotspot / Exons partially sequenced Chromosomal location
Size sequenced (bp)
B2M NM_004048 Hotspots exons 1 & 2, exon 3 15q21.1 360
BCL2 NM_000633 Hotspot exon 2 18q21.33 585
BRAF NM_004333 Exon 15 7q34 119
CARD11 NM_032415 Coiled-coil domain exons 4-9 7p22.2 1121
CD58 NM_001779 Exons 1-6 1p13.1 753
CD79A NM_001783 ITAM domain exons 4 & 5 19q13.2 183
CD79B NM_000626 ITAM domain exons 5 & 6 17q23.3 141
CDKN2A NM_000077+NM_058195+
NM_058197+NM_001195132
Exons 1, 2A, 2B, 3, 4 & 5 9p21.3 1,737
CDKN2B NM_004936 + NM_0078487 Exons 1A, 1B & 2 9p21.3 1289
CIITA NM_000246 Exons 1-19 16p13.13 3393
CREBBP NM_004380 Exons 1-31 16p13.3 7323
EP300 NM_001429 Exons 1-31 22q13.2 7245
EZH2 NM_004456 SET domain, hotspots exon 16 & 18 7q36.1 177 FOXO1 NM_002015 Hotspots exon 1 & FH domain exon 2 13q14.11 780
GNA13 NM_006572 Exons 1-4 17q24.1 1134
ID3 NM_002167 Exons 1 & 2 1p36.12 360
IRF4/MUM1 NM_002460 Exons 2-9 6p25.3 1356
ITPKB NM_002221 Exons 2-8 1q42.12 2830
KMT2D/MLL2 NM_003482 Exons 1-54 12q13.12 16614
MEF2B NM_001145785 Exons 2-9 19p13.11 1107
MFHAS1 NM_004225 Exons 1-3 8p23.1 3159
MYC NM_002467 Exons 1-3 8q24.21 1365
MYD88 NM_001172567 Exons 2-5 3p22.2 587
NOTCH1 NM_017617 PEST domain exon 34 9q34.3 1488
NOTCH2 NM_024408 Exons 26-28 & 34 (HD/PEST domains) 1p12-p11.2 2091
PIM1 NM_002648 Exons 1-6 6p21.2 942
PRDM1/BLIMP1 NM_001198 Exons 1-7 6q21 2478
SOCS1 NM_003745 Exon 2 16p13.13 636
STAT6 NM_001178078 Exons 9-14 (DNA binding domain hotspot) 12q13.3 795 TCF3 NM_001136139 B-HLH domain of E47 isoform exons 17 & 18 19p13.3 370
TNFAIP3 NM_006290 Exons 2-9 6q23.3 2373
TNFRSF14 NM_003820 Exons 1-8 1p36.32 852
TP53 NM_000546 Mutation hotspots exons 4-10 17p13.1 1004
XPO1 NM_003400 Exons 15-18 2p15 640
B. PGM Data analysis
Torrent Suite™ version 4.0 (Life Technologies) software was used to perform primary analysis, including signal processing, base calling, sequence alignment to the reference genome (hg19) and generation of Binary Alignment/Map (BAM) files. BAM files were used by Torrent Suite™’s Variant Caller to detect point mutations as well as short insertions and deletions using the “Somatic” and “Low Stringency”
default parameters. VCF files generated by Variant Caller were annotated by ANNOVAR(1).
Data generated from tumor DNA samples were considered of sufficient quality when more than 90% of targeted bases were read at least 20 times with sequencing and mapping precisions of at least Q20. Only frameshift deletions and insertions, nonframeshift deletions and substitutions, splicing, nonsynonymous, stopgain or stoploss Single Nucleotide Variations (SNVs) were kept. Variants with a minimal Variant Allele Frequency (MAF) greater than 1% in the 1000 genome database were considered as polymorphisms and were discarded(2). A normal probability plot defined thresholds separating true positives [confirmed by Sanger sequencing, TVC (Torrent variant calling) quality score ≥ 22] from true negatives (discredited by Sanger sequencing, TVC score < 9.5) and highlighted a gray zone (9.5 ≤ TVC
score < 22) in which variants must be confirmed by Sanger sequencing or pyrosequencing..
Further verification by Sanger sequencing was performed using a BigDye® Terminator v3.1 Cycle Sequencing Kit (Life Technologies) and an ABI PRISM 3130 analyzer (Life Technologies). Further verification by pyrosequencing was performed using the PyroMark PCR kit (Qiagen, France) with internal and sequencing primers designed using PyroMark software (Qiagen).
For circulating DNA variant calling, the parameter file was modified as follows:
hotspot_min_variant_score: 3; hotspot_strand_bias: 1; downsample_to_coverage: 30 000. In addition, a dedicated hotspot VCF file generated from variants found in tumor DNA was used. This file instructs the Variant Caller to include these positions in its output files, including evidence for a variant and the filtering thresholds that disqualify a variant candidate.
References
1. Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants from high- throughput sequencing data. Nucleic acids research. 2010 Sep;38(16):e164.
2. Abecasis GR, Auton A, Brooks LD, DePristo MA, Durbin RM, Handsaker RE, et al. An integrated map of genetic variation from 1,092 human genomes. Nature. 2012 Nov 1;491(7422):56-65.
Relevant reads sequenced Tumor: 885 Plasma: 21,078
Relevant reads sequenced Tumor: 582 Plasma: 18,827 Relevant reads sequenced Tumor: 553 Plasma: 4,685
1
Relevant reads sequenced Tumor: 1,381 Plasma: 51,195 Relevant reads sequenced Tumor: 1,263 Plasma: 21,661
1
t reads sequenced 1,886 70,466
1
Relevant reads sequenced Tumor: 3421 Plasma: 77,872
1 Relevant reads sequenced Tumor: 636 Plasma: 386
ant reads sequenced or: 3,724 a: 36,037 levant reads sequenced mor: 1,811 ma: 58,131
1
vant reads sequenced or: 2,041 ma: 85,369 levant reads sequenced mor: 1,852 ma: 50,862
1
