CN113604537A

CN113604537A - Method for constructing trace frozen tissue ATAC-seq sequencing library

Info

Publication number: CN113604537A
Application number: CN202110761466.3A
Authority: CN
Inventors: 姜昊; 邓昭敏
Original assignee: Sichuan Plo Hales Biotechnology Co ltd
Current assignee: Sichuan Plo Hales Biotechnology Co ltd
Priority date: 2021-07-06
Filing date: 2021-07-06
Publication date: 2021-11-05

Abstract

The invention provides a construction method of a trace frozen tissue ATAC-seq sequencing library, which comprises the following steps: s1: grinding the tissue, preparing cell suspension and extracting cell nucleus; s2: performing transposition and purification; s3: PCR enrichment is carried out, and a library is constructed and the fragment is screened; s4: and performing library quality inspection. The construction method of the trace frozen tissue ATAC-seq sequencing library has the following advantages: the method can well solve the problem that the existing ATAC-seq technology is not suitable for freezing the micro-tissues, especially the micro-tissues of the frozen squamous cell carcinoma.

Description

Method for constructing trace frozen tissue ATAC-seq sequencing library

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a construction method of a trace frozen tissue sample ATAC-seq sequencing library.

Background

Chromatin transposase accessibility sequencing (ATAC-seq) is a new technology of chromatin transposase accessibility based on high-throughput sequencing. Was first published in Nature Methods in 2013 and was developed by William JGreenleaf project group, university of stanford. The principle is to sequence the DNA sequence captured by the Tn5 enzyme by virtue of the property of transposase Tn5 to cleave the open chromatin region. Compared with the traditional chromatin opening research technology, the ATAC-seq sample has the advantages of small demand, simpler process and higher efficiency, and can capture the chromatin opening area from a small amount of cells by only two steps. This method can simultaneously obtain the location of "open" chromatin, map whole genome chromatin accessibility, predict transcription factor binding sites, determine the location of nucleosomes, and can also be used to determine chromatin accessibility in individual cells, thereby revealing heterogeneity within a population of cells. ATAC-seq has become the first technical method for studying chromatin openness in genomics. In the medical field, ATAC-seq is applied to drawing chromatin accessibility maps of complex diseases such as human tumors, autoimmune diseases, metabolic diseases, degenerative diseases and the like. The interaction of gene regulation is explored, the regulation mutation driving the occurrence and development of diseases is revealed, and the research of epigenetic inheritance of complex diseases is promoted.

Although the existing ATAC-seq technology makes up the defects of the prior art, the existing ATAC-seq technology still has the problems that the extraction efficiency of frozen tissue DNA is low, a DNA sequence in an open state cannot be obtained from a trace sample (less than or equal to 10mg), the establishment of a library for squamous cell carcinoma is difficult, and the like. Clinically, the cases that the important and trace tissues obstruct the clinical scientific exploration due to the insufficient sample size are all the current research hotspots and problems such as tumor heterogeneity research, tumor evolution process related research, flow type cell subset research, cell differentiation and the like.

In summary, the existing ATAC-seq technology is not suitable for freezing micro-tissues, especially for freezing squamous cell carcinoma micro-tissues. Therefore, a method for constructing a trace frozen tissue ATAC-seq sequencing library is provided to solve the above problems.

Disclosure of Invention

The invention aims to provide a method for constructing a trace frozen tissue ATAC-seq sequencing library aiming at the defects of the prior art, and the method for constructing the trace frozen tissue ATAC-seq sequencing library can well solve the problems.

In order to meet the requirements, the technical scheme adopted by the invention is as follows: the method for constructing the trace frozen tissue ATAC-seq sequencing library comprises the following steps:

s1: grinding the tissue, preparing cell suspension and extracting cell nucleus;

s2: performing transposition and purification;

s3: PCR enrichment is carried out, and a library is constructed and the fragment is screened;

s4: and performing library quality inspection.

The construction method of the trace frozen tissue ATAC-seq sequencing library has the following advantages:

the method can well solve the problem that the existing ATAC-seq technology is not suitable for freezing the micro-tissues, especially the micro-tissues of the frozen squamous cell carcinoma.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:

FIG. 1 schematically shows a flow diagram of a method for constructing a micro-frozen tissue ATAC-seq sequencing library according to one embodiment of the present application.

FIG. 2 schematically shows a library quality control diagram of frozen 3.5mg esophageal squamous carcinoma sample DNA obtained by a construction method of a micro-frozen tissue ATAC-seq sequencing library according to an embodiment of the present application.

FIG. 3 schematically shows a library quality inspection diagram of frozen 4.5mg esophageal squamous carcinoma sample DNA obtained by enteroscopy clamping according to the construction method of the trace frozen tissue ATAC-seq sequencing library in one embodiment of the application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the accompanying drawings and specific embodiments.

In the following description, references to "one embodiment," "an embodiment," "one example," "an example," etc., indicate that the embodiment or example so described may include a particular feature, structure, characteristic, property, element, or limitation, but every embodiment or example does not necessarily include the particular feature, structure, characteristic, property, element, or limitation. Moreover, repeated use of the phrase "in accordance with an embodiment of the present application" although it may possibly refer to the same embodiment, does not necessarily refer to the same embodiment.

Certain features that are well known to those skilled in the art have been omitted from the following description for the sake of simplicity.

According to one embodiment of the application, a method for constructing a micro-frozen tissue ATAC-seq sequencing library is provided, which comprises the following steps:

s1: grinding the tissue, preparing cell suspension and extracting cell nucleus;

s2: performing transposition and purification;

s4: and performing library quality inspection.

The steps are specifically described as follows:

the method of step S1 is: the micro-frozen tissue (about 3-5mg) was rapidly placed in a 2ml glass grinder (Kimblechase, 885301-; after grinding the tissue about 10-15 times using a grind bar a, the solution in this step was filtered through a cell filter screen into a new 50mL centrifuge tube; grinding with grinding rod B for about 20 times, filtering the solution with cell filter sieve, centrifuging at 4 deg.C and 350RCF for 5 min; absorbing and removing the supernatant, adding 350L of precooled 1xHB, and re-suspending and uniformly mixing; adding 400 mu L of 50% Iodixanol Solution, and uniformly mixing; add 600. mu.L of 30% Iodixanol Solution Iodixanol carefully under the 25% Solution layer; add 600 μ L of 40% iodixanol Solution carefully under the 30% Solution layer; the brake of the refrigerated centrifuge was set at 0, 4 ℃ and 3000RCF, and centrifugation was carried out for 20 min. Sucking the cell nucleus layer solution, adding a proper amount of precooled ATAC-RSB-Tween for dilution, and counting trypan blue; 8000 nuclei were counted, added to 1mL of pre-cooled ATAC-RSB-Tween solution and mixed well.

In step S2, the transposition reaction is specifically performed by: adding the cell nucleus extracted in the step (1) into a transposition reaction system containing 5 XTTBL 10 mu L, TTEMix V505 mu L, ddH2O 17.5.5 mu L and PBS 16.5 mu L, and placing the transposition reaction system in a PCR instrument to react for 30min at 37 ℃.

The purification process in step S2 is: 250 μ l (5 volumes of product) of PB buffer was added using Qiagen MinElute PCR purification Kit (250) and mixed well. Transferring into Column, standing at room temperature for 5min, then 8000rpm, centrifuging at room temperature for 1min. The collection tube was discarded and 750. mu.l of PE buffer was added to the Column, centrifuged at 13000rpm for 1min at room temperature. The liquid in the collection tube was discarded and centrifuged at 10000rpm for 1min at room temperature. Transfer Column to a new 1.5ml Lo-Bind centrifuge tube, uncap and stand for 3 min. Add 11. mu.l ddH2O, drop-add onto membrane, let stand at room temperature for 5min, centrifuge at room temperature at 10000rpm for 1min.

The specific operation of PCR enrichment in step S3 is as follows: and (3) preparing a 50 mu L reaction system by taking the DNA product purified in the step (2) as a template: transposed DNA 10. mu.l, 5 XPCR MIX 25. mu.l, N5XX 5. mu.l, N7XX 5. mu.l, ddH2O 5. mu.l, mixed and amplified according to the following conditions: extension at 72 ℃ for 5min, pre-denaturation at 98 ℃ for 30sec, followed by amplification for 5 cycles with the following parameters: denaturation at 98 ℃ for 10 sec, denaturation at 63 ℃ for 30sec, extension at 72 ℃ for 1min, and finally Hold at 12 ℃.

The number of minimal cycle PCR amplifications was determined using qPCR, quantitative PCR (qPCR)15 μ l reaction: mu.l of the DNA amplified 5 times as described above, 0.06. mu.l of 100 XSSYBR Green I, 5. mu.l of 2X 2PCR MIX, 0.25. mu.l of N5XX 0.25, 0.25. mu.l of N7XX 0.25, 4.44. mu.l of ddH2O 4.44, the same as described above. After being mixed uniformly, the mixture is prepared according to the following conditions: 98 ℃ for 30 seconds, 20 cycles: 10 seconds at 98 ℃, 30 seconds at 63 ℃ and 1 minute at 72 ℃.

The PCR cycles that need to be added were predicted from the above qPCR results (1/3Rn max), and the remaining 45. mu.l sample was PCR amplified according to the following procedure: pre-denaturation at 98 ℃ for 30 seconds, followed by amplification for 5 cycles with the following parameters: denaturation at 98 ℃ for 10 sec, denaturation at 63 ℃ for 30sec, extension at 72 ℃ for 1min, and finally Hold at 12 ℃.

The amplification products in step S3 are subjected to length sorting: adding 50 mu L of DNA, and carrying out two rounds of bead sorting according to the volume ratio of the DNA to the Beads of 1:0.55 and 1:1 respectively to obtain the DNA with the target size.

Concentrations were measured using qubit4.0 and fragment distribution detection was performed using PART8 using Qsep 1.

Example 1:

a method for establishing a library by freezing trace esophageal squamous carcinoma tissue ATAC-seq sequencing.

(1) Reagent for configuring ATAC-seq library construction

a.6X HB unstable

b.1X HB unstable

c.50％Iodixanol Solution

Reagent	Volume of 1 sample
		6X HB unstable	75
60％Iodixanol Solution	375

d.40％Iodixanol Solution

Reagent	Volume of 1 sample
		6X HB unstable	112.5
1M Sucrose	108
		60％Iodixanol Solution	450
H2O	4.5

e.30％Iodixanol Solution

Reagent	Volume of 1 sample
		6X HB unstable	112.5
1M Sucrose	108
		60％Iodixanol Solution	337.5
H2O	117

(2) Grinding the tissue to extract nuclei

a. Draw pre-cooled 2mL 1XHB unstable buffer into the tissue grinder, place on ice, and stand by.

b. The micro esophageal squamous carcinoma specimen which is nipped under the endoscope is quickly weighed, and the weight is 5 mg.

c. Quickly putting into a tissue grinder containing HB unstable buffer, standing and thawing for about 5min on ice.

d. Grinding the substrate for about 10 to 15 times by using a grinding rod A.

e. The solution from the previous step was filtered through a cell filter screen into a new 50mL centrifuge tube.

f. And then ground about 15 times using a B grinding rod.

g. The solution from the previous step was filtered through a cell filter screen into a new 50mL centrifuge tube.

h. Transfer to a new 2mL Lo-Bind centrifuge tube, centrifuge at 350RCF for 5min at 4 ℃.

i. The supernatant was aspirated off to a volume of approximately 50L, 350L of pre-cooled 1xHB was added, and the mixture was resuspended and homogenized.

j. Add 400. mu.L of 50% Iodixanol Solution and mix well.

k. Add 600. mu.L of a 30% Solution of Iodixanol Solution Iodixanol carefully below the 25% Solution layer.

mu.L of a 40% Solution of iodixanol was carefully added under the 30% Solution layer.

m. setting the brake of the refrigerated centrifuge at 0, 4 ℃ and 3000RCF, and centrifuging for 20 min.

n. a clear nuclear layer was observed, the appropriate amount of supernatant solution was aspirated off, and the nuclear layer solution was pipetted into a new 1.5mL Lobind centrifuge tube.

Add about 200 μ L of pre-cooled ATAC-RSB-Tween to dilute and mix well.

And p, sucking 5 mu L of the solution in the previous step, mixing the solution with 5 mu L of trypan blue solution, counting the mixture on a blood counting plate, and calculating the concentration of cell nuclei.

q. transferring 6000 cell nucleuses to a new 1.5mL Lo-Bind centrifuge tube, adding 1mL precooled ATAC-RSB-Tween solution, mixing uniformly, carrying out 500RCF at 4 ℃, and centrifuging for 10 min.

(3) Rotating seat

a. Tn5 interaction mix was prepared as follows:

components	1 sample
		NF H2O	17.5μL
PBS	16.5μL
		5X TTBL	10μL
Digitonin	0.5μL
		Tween-20	0.5μL
TTE-Mix V50	5μL

b. Discarding the supernatant, sucking 50. mu.L of mix prepared in the previous step into an EP tube, and mixing well

c.37 ℃ incubation for 30min

(4) Purification of

a. 250 μ l (5 volumes of product) of PB buffer was added using Qiagen MinElute PCR purification Kit (250) and mixed well.

b. Transferring into Column, standing at room temperature for 5min, then 8000rpm, centrifuging at room temperature for 1min.

c. The collection tube was discarded and 750. mu.l of PE buffer was added to the Column, centrifuged at 13000rpm for 1min at room temperature.

d. The liquid in the collection tube was discarded and centrifuged at 10000rpm for 1min at room temperature.

e. Transfer Column to a new 1.5ml Lo-Bind centrifuge tube, uncap and stand for 3 min.

f. Add 11. mu.l ddH2O, drop-add onto membrane, let stand at room temperature for 5min, centrifuge at room temperature at 10000rpm for 1min.

(1) PCR enrichment and library construction.

a. Configuring a PCR reaction system

Reagent	Volume of
		Transferred DNA	10μl
5X PCR MIX	25μl
		N5XX*	5μl
N7XX*	5μl
		NF water	5μl
Total of	50μl

PCR reaction procedure

c. The qPCR system was configured as follows

qPCR reaction procedure

e. The number of cycles to be added was predicted from the above results (1/3Rn max), and the remaining 45. mu.l sample was PCR-amplified according to the following table procedure

(5) Library fragment screening

a. Vortex and mix VAHTS DNAclean Beads and suck 27.5. mu.l volume to 50. mu.l PCR product, use the pipettor to blow 10 times to mix well, room temperature incubation 5 min.

b. The reaction tube was centrifuged briefly and placed on a magnetic stand to separate the magnetic beads from the liquid, after the solution was clarified (about 5min), the supernatant was carefully transferred to a fresh sterile PCR tube and the magnetic beads discarded.

c. Vortex and mix VAHTS DNAclean Beads and suck 50 u l volume to supernatant, vortex or use the pipettor to blow 10 times fully mix, room temperature incubation for 5 min.

d. The reaction tube was briefly centrifuged and placed on a magnetic stand to separate the magnetic beads from the liquid, and the supernatant carefully removed after the solution cleared (about 5 min).

e. The reaction tube was kept on the magnetic frame all the time, and 200. mu.l of freshly prepared 80% ethanol was added to rinse the beads. Incubate at room temperature for 30sec and carefully remove the supernatant. Rinse 2 times.

f. Keeping the reaction tube on the magnetic frame all the time, and opening the cover to dry the magnetic beads in air for about 5 min.

g. The reaction tube was removed from the magnetic holder and eluted with 22. mu.l of sterilized ultrapure water. Vortexing or blowing and beating for 10 times by using a pipette, and fully mixing, and incubating for 5min at room temperature.

(5) Library quality inspection

Concentrations were measured using qubit4.0 and fragment distribution detection was performed using Qsep 1. As shown in FIG. 3, the size range of the library fragment is 200-700bp, and the main peaks are about 200bp, 500bp, 700bp, and have a sawtooth peak shape.

Example 2:

freezing a library building method for sequencing of micro intestinal tissue ATAC-seq.

(1) Reagent for configuring ATAC-seq library construction

a.6X HB unstable

b.1X HB unstable

c.50％Iodixanol Solution

Reagent	Volume of 1 sample
		6X HB unstable	75
60％Iodixanol Solution	375

d.40％Iodixanol Solution

e.30％Iodixanol Solution

(2) Grinding the tissue to extract nuclei

d. About 20 grindings were performed using an a grinding rod.

f. And then ground about 20 times using a B grinding rod.

h. Transfer to a new 2mL Lo-Bind centrifuge tube, centrifuge at 350RCF for 5min at 4 ℃. The supernatant was aspirated off to a volume of approximately 50L, 350L of pre-cooled 1xHB was added, and the mixture was resuspended and homogenized.

i. Add 400. mu.L of 50% Iodixanol Solution and mix well.

j. Add 600. mu.L of a 30% Solution of Iodixanol Solution Iodixanol carefully below the 25% Solution layer.

k. 600 μ L of a 40% Solution of iodixanol Solution in iodixanol was carefully added under the 30% Solution layer.

l, setting the brake of the refrigerated centrifuge at 0, 4 ℃ and 3000RCF, and centrifuging for 20 min.

m. a clear nuclear layer was observed, the appropriate amount of supernatant solution was aspirated off, and the nuclear layer solution was pipetted into a new 1.5mL Lobind centrifuge tube.

n. adding about 200. mu.L of precooled ATAC-RSB-Tween for dilution and mixing evenly.

o. aspirate 5 μ L of the solution from the previous step and mix with 5 μ L of trypan blue solution, count on a hemocytometer, and calculate the concentration of nuclei.

p. transfer 30000 nuclei to a new 1.5mL Lo-Bind centrifuge tube, add 1mL pre-chilled ATAC-RSB-Tween solution, mix well, centrifuge at 4 ℃ 500RCF for 10 min.

(3) Rotating seat

a. Tn5 interaction mix was prepared as follows:

c.37 ℃ incubation for 30min

(4) Purification of

(5) PCR enrichment and library construction.

a. Configuring a PCR reaction system

Procedure for PCR reaction

g. The qPCR system was configured as follows

h.qpcr reaction procedure

i. The number of cycles to be added was predicted from the above results (1/3Rn max), and the remaining 45. mu.l sample was PCR-amplified according to the following table procedure

(5) Library fragment screening

a. VAHTS DNA Clean Beads were vortexed and pipetted to 50. mu.l volume of PCR product in 27.5. mu.l volume, pipetted 10 times to mix well, and incubated at room temperature for 5 min.

c. Vortex and mix VAHTS DNA Clean Beads and suck 50 ul volume to supernatant, vortex or use the pipettor to blow 10 times fully mix, room temperature incubation 5 min.

(5) Library quality inspection

Concentrations were measured using qubit4.0 and fragment distribution detection was performed using Qsep 1. As shown in FIG. 3, the size range of the library fragment is 200-700bp, and the main peaks are about 200bp, 300bp, and 500bp, which are in the shape of sawtooth peaks.

The above-mentioned embodiments only show some embodiments of the present invention, and the description thereof is more specific and detailed, but should not be construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the claims.

Claims

1. A method for constructing a trace frozen tissue ATAC-seq sequencing library is characterized by comprising the following steps:

s1: grinding the tissue, preparing cell suspension and extracting cell nucleus;

s2: performing transposition and purification;

s4: and performing library quality inspection.

2. The method for constructing the ATAC-seq sequencing library of micro-frozen tissue according to claim 1, wherein S1: the steps of grinding the tissue, preparing a cell suspension and extracting nuclei include:

placing the micro-frozen tissue into a 2ml glass grinder containing 1X HB unstable buffer, and standing for 5 minutes on ice;

after grinding the tissue about 10-15 times using a first grinding rod, the solution in this step was filtered through a cell filter screen into a new 50mL centrifuge tube; grinding with a second grinding rod for about 20 times, filtering the solution with cell filter sieve, and centrifuging at 4 deg.C and 350RCF for 5 min;

absorbing and removing the supernatant, adding 350L of precooled 1xHB, and re-suspending and uniformly mixing; adding 400 mu L of 50% Iodixanol Solution, and uniformly mixing;

add 600. mu.L of a 30% Iodixanol Solution of Iodixanol under the 25% Solution layer;

600 μ L of a 40% Solution of iodixanol Solution in iodixanol was added under the 30% Solution layer;

setting the brake of a refrigerated centrifuge to be 0, centrifuging for 20min at 4 ℃ under 3000RCF environment, sucking a cell nucleus layer solution, adding a proper amount of precooled ATAC-RSB-Tween for dilution, and counting trypan blue;

8000 nuclei were counted, added to 1mL of pre-chilled ATAC-RSB-Tween solution and mixed well.

3. The method for constructing a micro frozen tissue ATAC-seq sequencing library according to claim 1, wherein the steps of transposition and purification are as follows:

the nuclei extracted in step S1 were added to a transposition reaction system containing 5 XTTBL 10. mu.L, TTEMix V505. mu.L, ddH2O 17.5.5. mu.L, PBS 16.5. mu.L, placed in a PCR apparatus and reacted at 37 ℃ for 30 min.

4. The method for constructing a micro frozen tissue ATAC-seq sequencing library according to claim 1, wherein the steps of transposing and purifying comprise the following steps:

add 250. mu.l PB buffer to mix using Qiagen MinElute PCR purification Kit (250);

transferring into Column, standing at room temperature for 5min, and centrifuging at 8000rpm at room temperature for 1 min;

discarding the liquid in the collection tube, adding 750 μ l of PE buffer into Column, and centrifuging at 13000rpm for 1 min;

discarding liquid in the collecting pipe, and centrifuging for 1min at room temperature under 10000 rpm;

transferring the Column into a new 1.5ml Lo-Bind centrifuge tube, opening a cover and standing for 3 min;

add 11. mu.l ddH2O, drop-add onto membrane, stand 5min at room temperature, and centrifuge for 1min at room temperature 10000 rpm.

5. The method for constructing a micro frozen tissue ATAC-seq sequencing library according to claim 1, wherein: the specific steps for PCR enrichment were as follows:

using the DNA product purified in step S2 as a template, a 50. mu.L reaction system was prepared:

transposed DNA 10. mu.l, 5 XPCR MIX 25. mu.l, N5XX 5. mu.l, N7XX 5. mu.l, ddH2O 5. mu.l, mixed and amplified according to the following conditions: extension at 72 ℃ for 5min and pre-denaturation at 98 ℃ for 30 sec;

amplification was performed for 5 cycles with the following parameters: denaturation at 98 ℃ for 10 sec, denaturation at 63 ℃ for 30sec, extension at 72 ℃ for 1min, and finally Hold at 12 ℃.

6. The method for constructing the ATAC-seq sequencing library of micro-frozen tissue as claimed in claim 5, wherein the number of minimum cycle PCR amplifications is determined by qPCR, and the quantitative PCR (qPCR)15 μ l reaction system is as follows:

5 μ l of DNA amplified 5 times, 0.06 μ l of 100 XSSYBR Green I, 5 μ l of 2X 2PCR MIX, 0.25 μ l of the same as described above for N5XX 0.25, N7XX 0.25.25 μ l, ddH2O 4.44.44 μ l;

after mixing uniformly, the method comprises the following steps: 98 ℃ for 30 seconds, 20 cycles: 10 seconds at 98 ℃, 30 seconds at 63 ℃ and 1 minute at 72 ℃.

7. The method for constructing ATAC-seq sequencing library of micro-frozen tissue according to claim 6, wherein: the PCR cycles that need to be added are predicted from the qPCR results (1/3Rn max), and the remaining 45. mu.l of sample is PCR amplified according to the following procedure: pre-denaturation at 98 ℃ for 30 seconds, followed by amplification for 5 cycles with the following parameters: denaturation at 98 ℃ for 10 sec, denaturation at 63 ℃ for 30sec, extension at 72 ℃ for 1min, and finally Hold at 12 ℃.

8. The method for constructing a micro frozen tissue ATAC-seq sequencing library according to claim 7, wherein: the process of PCR enrichment, library construction and length sorting of the amplified products in the fragment screening step is as follows: adding 50 mu L of DNA, and carrying out two rounds of bead sorting according to the volume ratio of the DNA to the Beads of 1:0.55 and 1:1 respectively to obtain the DNA with the target size.

9. The method for constructing the ATAC-seq sequencing library of micro-frozen tissue according to claim 8, wherein the method comprises the following steps: concentrations were measured using qubit4.0 and fragment distribution detection was performed using Qsep 1.