Step 1: Sample processing and gDNA extraction Step 2: Libraries

Step 1: Sample processing and gDNA extraction 5 MCL-patients from FIL MCL0208 trial

Step 2: Libraries preparation

Step 3: Sequencing and FASTQ quality check

Input 500 ng DNA/sample

Sample tissue Blood sample or Bone marrow sample

1st Round PCR VDJ

aagggttttcccc

Follow-up samples from MCL 0208 trial

Screening primers: Diagnostic sample IGH FR1 Dilution in BC 10-1 10-2

FU2

FU3

Controls/ sequence run

10-1

1-3 1-4

Step 4: HashClone analysis

AtDI A

FU3 FU1

Dilution in BC

Artificial FUs

Pat i ent i

1st Round PCR

Follow-up samples from MCL 0208 trial FU1 sampleFU2 Diagnostic

ccgggttaacctt

Si gni ◆cantkmeri dent i ◆cat i on HashCheckerFreq

k-mers1

FU2

HashCheckerFreq

k-mers2

FU3

HashCheckerFreq

k-mers3

FUn

HashCheckerFreq

k-mers3

10-2 10-3 1-4 Gener at i onofr eadsi gnat ur es HashCheckerSignature

Artificial FUs

CompCheckerKme

Step 5: Filtered strategy for B-cell clones selection Phase A: Clone selection in diagnostic samples with reads Hela frequency higher than 5%

Buffy coat

Si gni ⇧cant k mer

Char act er i sat i onand eval uat i onoft hecancercl ones

Si gnat ur e1

HashCheckerSignature

Si gnat ur e2

HashCheckerSignature

Si gnat ur es3

HashCheckerSignature

Si gnat ur en

CompCheckerRead

Cl ones

Phase B: IMGT software: HashClone-VDJ sequences aligment to IGH reference database to check sequence homology.

Step 6: MRD monitoring

:

Fi gur e1-HashCl onepi pel i ne

FU = (Q-PCR value)

Dia (Q-PCR value)

x

FU (NGS reads)

Dia (NGS reads)