Supplemental Digital Content 1. Next Generation Sequencing Strategy and
Method.
Design of T-Cell Lymphoma Panel (TCLP)
A next generation sequencing mutation panel was designed to target hotspot mutations
in 38 genes on the JAK/STAT or other signaling pathways or previously found to be
mutated in T-cell lymphomas using AmpliSeq Designer (Life Technologies). Two
custom primer pools contained a total of 227 amplicons.
DNA Samples
The paraffin-embedded tissue sections were macrodissected to enrich to at least 20%
tumor content. DNA was extracted using the Qiagen QIAamp DNA FFPE Tissue Kit on
a QIAcube according to the instructions of the manufacturer.
Targeted Amplicon Library Preparation
AmpliSeq PCR reaction was conducted with Ion AmpliSeq Library Kit 2.0 in a total
volume of 10 ul containing 2 ul 5x Ion AmpliSeq HiFi Mix, 5 ul 2x TCLP primer pool, and
12 ng gDNA template. Two reactions were run with separate primer pools for each DNA
sample for 22-24 cycles. Sequencing libraries were prepared from the combined
amplified DNA by partial digestion of primer sequences, ligation of adapters, and
assignment of unique barcodes. Libraries were quantitated using a ViiA7 real-time PCR
system.
Emulsion PCR and Sequencing
Four libraries were pooled together and placed into an Ion Chef system, which
automates emulsion PCR and chip loading steps for subsequent NGS sequencing. The
sequencing was performed on the PGM with the Ion PGM Sequencing 200 v2 IC Kit.
Data Analysis
Mutation analysis was carried out with Ion variantCaller (v4.2), and confirmed using
Integrative Genomics Viewer (IGV). The variant annotation and interpretation were
performed in cloud-based Ion Reporter (v4.2).
Supplemental Digital Content 2. Table of Genes Included in the Sequencing
Panel
BRAF
CCR4
CD247 (CD3 -zeta)
CD3D
CD3E
CD3G
DNMT3A
EZH2
FBXW7
FYN
GNB1
HRAS
IDH1
IDH2
IL6ST
IL7R
JAK1
JAK2
JAK3
KRAS
LCK
MYD88
NOTCH1
NRAS
PIK3CA
PIK3CD
PLCG1
PTPN1
PTPN2
RHOA
SOCS1
STAT3
STAT5A
STAT5B
SYK
TET2
TYK2
ZAP70