Fragmentation of target molecules for sequencing

Patent metadata

Field	Value
Publication number	US-2021379591-A1
Application number	US-202117236826-A
Country	US
Kind code	A1
Filing date	Apr 21, 2021
Priority date	Apr 22, 2020
Publication date	Dec 9, 2021
Grant date	—

Abstract

Methods and devices for preparing target molecules (e.g., target nucleic acids or target proteins) from a biological sample are provided herein. In some embodiments, methods and devices involve sample lysis, sample fragmentation, enrichment of target molecule(s), and/or functionalization of target molecule(s).

First claim

What is claimed is:

1 . A device for preparing a biological sample for sequencing, wherein the device comprises an automated module configured to receive (iii) a fragmentation cartridge comprising one or more microfluidic channels and configured to digest or fragment at least one of the one or more target molecules to produce a fragmented sample; and one or more of the cartridges selected from (i) a lysis cartridge (ii) an enrichment cartridge, and (iv) a functionalization cartridge; wherein (i), (ii), and (iv) are defined as follows:
(i) a lysis cartridge comprises one or more microfluidic channels and configured to intake a biological sample comprising one or more target molecules and produce a lysed sample; (ii) an enrichment cartridge comprises one or more microfluidic channels and is configured to enrich at least one of the one or more target molecules to produce an enriched sample; and (iv) a functionalization cartridge comprises one or more microfluidic channels and is configured to functionalize a terminal moiety of at least one of the one or more target molecules to produce a functionalized sample.

2 . The device of claim 1 , wherein the biological sample is a single cell, mammalian cell tissue, animal sample, fungal sample, plant sample, blood sample, saliva sample, sputum sample, fecal sample, urine sample, buccal swab sample, amniotic sample, seminal sample, synovial sample, spinal sample, or pleural fluid sample.

3 . The device of claim 1 , wherein the one or more target molecules are nucleic acids or proteins.

4 . The device of claim 1 , wherein the one or more microfluidic channels are configured to contain and/or transport fluid(s) and/or reagent(s).

5 . The device of claim 1 , wherein the fragmentation cartridge comprises non-enzymatic reagents that digest or fragment the sample and/or the one or more target molecules.

6 . The device of claim 5 , wherein the non-enzymatic reagents that digest or fragment the sample and/or the one or more target molecules comprise detergents, acids, and/or bases.

7 . The device of claim 5 , wherein the non-enzymatic reagents that digest or fragment the sample and/or the one or more target molecules comprise cyanogen bromide, hydroxylamine, iodosobenzoic acid, dimethyl sulfoxide, hydrochloric acid, BNPS-skatole [2-(2-nitrophenylsulfenyl)-3-methylindole], and/or 2-nitro-5-thiocyanobenzoic acid.

8 . The device of claim 1 , wherein the fragmentation cartridge comprises one or more enzymatic reagents that digest or fragment at least one of the one or more target molecules.

9 . The device of claim 8 , wherein the one or more enzymatic reagents comprise one or more proteases, optionally wherein the one or more proteases are selected from the group consisting of: trypsin, chymotrypsin, LysC, LysN, AspN, GluC and ArgC.

10 . The device of claim 8 , wherein the one or more enzymatic reagents comprise one or more endonucleases or exonucleases.

11 . The device of claim 1 , wherein the fragmentation cartridge can be heated at an elevated temperature (e.g., 20-60° C.).

12 . The device of claim 1 , wherein the device is configured to heat the fragmentation cartridge at an elevated temperature (e.g., 20-60° C.).

13 . The device of claim 1 , wherein the module is further configured to receive a lysis cartridge, optionally wherein the lysis cartridge and the fragmentation cartridge are connected by one or more microfluidic channels.

14 . The device of claim 1 , wherein the fragmentation cartridge is positioned to receive the lysed sample from the lysis cartridge.

15 . The device of claim 13 , wherein the device is configured such that the lysed sample is enriched prior to fragmentation.

16 . A device for preparing one or more target molecules, configured to perform step (iii) fragment the one or more target molecules; and one or more of the following steps selected from (i), (ii), and (iv),
wherein (i), (ii), and (iv) are defined as follows:
(i) lyse a biological sample comprising one or more target molecules;
(ii) enrich at least one of the one or more target molecules and/or at least one non-target molecule; and
(iv) functionalize a terminal moiety of the one or more target molecules.

17 . The device of claim 16 , wherein one or more of the steps selected from (i), (ii), (iii), and (iv) are performed in a cartridge.

18 . The device of claim 16 , wherein the one or more steps are performed in the same cartridge.

19 . A method for preparing one or more target molecules, comprising step (iii) fragment the one or more target molecules; and one or more of the following steps selected from (i), (ii), and (iv),
wherein (i), (ii), and (iv) are defined as follows:
(i) lyse a biological sample comprising one or more target molecules
(ii) enrich at least one of the one or more target molecules and/or at least non-target molecule; and
(iv) functionalize a terminal moiety of the one or more fragmented target molecules;

wherein step (iii) is performed in an automated sample preparation device.

20 . A cartridge for preparing one or more target molecules, configured to perform step (iii) fragment the one or more target molecules; and one or more of the following steps selected from (i), (ii), and (iv),
wherein (i), (ii), and (iv) are defined as follows:
(i) lyse a biological sample comprising one or more target molecules
(ii) enrich at least one of the one or more target molecules and/or at least one non-target molecule; and
(iv) functionalize a terminal moiety of the one or more target molecules.

Assignees

• Quantum Si Inc

Inventors

• Rothberg Jonathan M
• Leamon John H
• Schultz Jonathan C
• Millham Michele
• Lv Caixia
• Huang Haidong
• Nani Roger
• Ad Omer
• Reed Brian
• Dyer Matthew
• Boer Robert E

Classifications

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• B01L2400/0481

  squeezing of channels or chambers · CPC title

• B01L2300/0893

  having a very large number of wells, microfabricated wells · CPC title

• B01L2200/16

  Reagents, handling or storing thereof · CPC title

• B01L2300/021

  Identification, e.g. bar codes · CPC title