Trimming: The number of base pairs that will be removed from either the 3' or the 5' end. In QIIME 2, you'll most commonly see trim-left (which will trim off the requested number of base pairs from the 5' end), but in qiime feature-classifier extract-reads, there is also a trim-right parameter, which refers to the 3' end.

Truncating: The position at which reads will be truncated (counting from the 5' end). Reads that are shorter than the value provided will be discarded. The command you'll see this action represented by within QIIME 2 is trunc-len. Something to note is that in the denoising pipelines within the QIIME 2 core framework, truncation occurs before trimming.

conda activate rescript

conda install -c conda-forge -c bioconda -c qiime2 -c https://packages.qiime2.org/qiime2/2023.5/tested/ -c defaults qiime2 q2cli q2templates q2-types q2-longitudinal q2-feature-classifier 'q2-types-genomics>2023.2' "pandas>=0.25.3" xmltodict ncbi-datasets-pylib

pip install git+https://ghproxy.com//https://github.com/bokulich-lab/RESCRIPt.git

创建COI库
qiime rescript get-ncbi-data --p-query '(cytochrome c oxidase subunit 1[Title] OR cytochrome c oxidase subunit I[Title] OR cytochrome oxidase subunit 1[Title] OR cytochrome oxidase subunit I[Title] OR COX1[Title] OR CO1[Title] OR COI[Title]) AND BARCODE[KYWD])' --verbose --output-dir NCBIdata_BOLDonly2 --o-sequences coi_genbank.qza --o-taxonomy coi_tax.qza

创建16s库

the forward- and reverse-reads from the sequencing were joined using the FLASH software (Magoc & Salzberg, 2011).

nanopore:
Reads were basecalled and demultiplexed using Guppy
The resulting FASTQ files were used as input of Porechop (Wick R., 2017) for adaptor removal and barcode de-multiplexing

FASTQ files were aligned against the NCBI-nr protein database (Nov. 2017) using DIAMOND v0.9.22 (blastx option) setting the -F option to 15, to consider frame-shift errors in the sequences and the – rangeCulling and –top options set to 10 to scan the whole sequence for alignments with a 10% of the best local bit score

The taxonomic binning of short reads from Illumina was performed using the daa2rma program from MEGAN Community Edition (CE) v6.11. with the option -a2t, to map the reads to the NCBI-taxonomy mapping file containing protein accessions

Relative taxonomic abundances were obtained for each samples and platform representing the number of reads assigned to each taxon.

Relative abundances, alpha (Shannon and Simpson indexes) and beta diversity -using Bray-Curtis dissimilarity-were analyzed using the phyloseq (McMurdie & Holmes, 2013), vegan (Oksanen et al., 2019), and microbiome R (Leo Lahti et al., 2012-2019) packages.

Short reads from the Illumina dataset were assembled using Megahit (Li, Liu, Luo, Sadakane, & Lam, 2015) with the default parameters using the joined forward and reverse reads.

The nanopore reads were assembled using Canu (Koren et al., 2017) setting the options minReadLength = 100, minOverlapLength = 100 as the resulting reads were too short for the default parameters (less than 10,000 bp), and genomeSize = 2.5m, as described in the documentation for metagenome assemblies.

The quality of the assemblies was assessed using Quast v4.0 (Mikheenko, Valin, Prjibelski, Saveliev, & Gurevich, 2016).

Rarefaction curves(稀疏曲线)

alpha diversity indexes

relative abundances

The core dataset from VFDB (setA), which is composed of genes associated with experimentally verified virulence factors (VFs) for 53 bacterial species

The VirulenceFinder dataset which includes virulence genes for Listeria, Staphylococcus aureus, Escherichia coli/Shigella and Enterococcus

A manually curated dataset of reference virulence genes for Escherichia coli (Coli_Ref).

这个工具把几个毒力鉴定的做了综合

You are now running subprogram: FixCircRef

WARNING @ [2022-05-24 12:32:34 UTC]: The output directory is not provided!
WARNING @ [2022-05-24 12:32:34 UTC]: Will mkdir /qap_Results_for_FixCircRef_20220524_12h32m34s and use it as the output directory.
INFO @ [2022-05-24 12:32:34 UTC]: Building Bowtie2 index for /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/HBV_C_AB033556.2line.fix0.fasta.
INFO @ [2022-05-24 12:32:34 UTC]: /opt/qap/bin/3rdPartyTools/bowtie2/bowtie2-build /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/HBV_C_AB033556.2line.fix0.fasta /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/HBV_C_AB033556.2line.fix0
Settings:
Output files: "/qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/HBV_C_AB033556.2line.fix0..bt2"
Line rate: 6 (line is 64 bytes)
Lines per side: 1 (side is 64 bytes)
Offset rate: 4 (one in 16)
FTable chars: 10
Strings: unpacked
Max bucket size: default
Max bucket size, sqrt multiplier: default
Max bucket size, len divisor: 4
Difference-cover sample period: 1024
Endianness: little
Actual local endianness: little
Sanity checking: disabled
Assertions: disabled
Random seed: 0
Sizeofs: void:8, int:4, long:8, size_t:8
Input files DNA, FASTA:
/qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/HBV_C_AB033556.2line.fix0.fasta
Building a SMALL index
Reading reference sizes
Time reading reference sizes: 00:00:00
Calculating joined length
Writing header
Reserving space for joined string
Joining reference sequences
Time to join reference sequences: 00:00:00
bmax according to bmaxDivN setting: 928
Using parameters --bmax 696 --dcv 1024
Doing ahead-of-time memory usage test
Passed! Constructing with these parameters: --bmax 696 --dcv 1024
Constructing suffix-array element generator
Building DifferenceCoverSample
Building sPrime
Building sPrimeOrder
V-Sorting samples
V-Sorting samples time: 00:00:00
Allocating rank array
Ranking v-sort output
Ranking v-sort output time: 00:00:00
Invoking Larsson-Sadakane on ranks
Invoking Larsson-Sadakane on ranks time: 00:00:00
Sanity-checking and returning
Building samples
Reserving space for 12 sample suffixes
Generating random suffixes
QSorting 12 sample offsets, eliminating duplicates
QSorting sample offsets, eliminating duplicates time: 00:00:00
Multikey QSorting 12 samples
(Using difference cover)
Multikey QSorting samples time: 00:00:00
Calculating bucket sizes
Splitting and merging
Splitting and merging time: 00:00:00
Split 2, merged 4; iterating...
Splitting and merging
Splitting and merging time: 00:00:00
Avg bucket size: 463.5 (target: 695)
Converting suffix-array elements to index image
Allocating ftab, absorbFtab
Entering Ebwt loop
Getting block 1 of 8
Reserving size (696) for bucket 1
Calculating Z arrays for bucket 1
Entering block accumulator loop for bucket 1:
bucket 1: 10%
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Sorting block of length 354 for bucket 1
(Using difference cover)
Sorting block time: 00:00:00
Returning block of 355 for bucket 1
Getting block 2 of 8
Reserving size (696) for bucket 2
Calculating Z arrays for bucket 2
Entering block accumulator loop for bucket 2:
bucket 2: 10%
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Sorting block of length 521 for bucket 2
(Using difference cover)
Sorting block time: 00:00:00
Returning block of 522 for bucket 2
Getting block 3 of 8
Reserving size (696) for bucket 3
Calculating Z arrays for bucket 3
Entering block accumulator loop for bucket 3:
bucket 3: 10%
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Sorting block of length 409 for bucket 3
(Using difference cover)
Sorting block time: 00:00:00
Returning block of 410 for bucket 3
Getting block 4 of 8
Reserving size (696) for bucket 4
Calculating Z arrays for bucket 4
Entering block accumulator loop for bucket 4:
bucket 4: 10%
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Sorting block of length 291 for bucket 4
(Using difference cover)
Sorting block time: 00:00:00
Returning block of 292 for bucket 4
Getting block 5 of 8
Reserving size (696) for bucket 5
Calculating Z arrays for bucket 5
Entering block accumulator loop for bucket 5:
bucket 5: 10%
bucket 5: 20%
bucket 5: 30%
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Sorting block of length 579 for bucket 5
(Using difference cover)
Sorting block time: 00:00:00
Returning block of 580 for bucket 5
Getting block 6 of 8
Reserving size (696) for bucket 6
Calculating Z arrays for bucket 6
Entering block accumulator loop for bucket 6:
bucket 6: 10%
bucket 6: 20%
bucket 6: 30%
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Sorting block of length 670 for bucket 6
(Using difference cover)
Sorting block time: 00:00:00
Returning block of 671 for bucket 6
Getting block 7 of 8
Reserving size (696) for bucket 7
Calculating Z arrays for bucket 7
Entering block accumulator loop for bucket 7:
bucket 7: 10%
bucket 7: 20%
bucket 7: 30%
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Sorting block of length 659 for bucket 7
(Using difference cover)
Sorting block time: 00:00:00
Returning block of 660 for bucket 7
Getting block 8 of 8
Reserving size (696) for bucket 8
Calculating Z arrays for bucket 8
Entering block accumulator loop for bucket 8:
bucket 8: 10%
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Sorting block of length 225 for bucket 8
(Using difference cover)
Sorting block time: 00:00:00
Returning block of 226 for bucket 8
Exited Ebwt loop
fchr[A]: 0
fchr[C]: 832
fchr[G]: 1857
fchr[T]: 2662
fchr[$]: 3715
Exiting Ebwt::buildToDisk()
Returning from initFromVector
Wrote 4195809 bytes to primary EBWT file: /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/HBV_C_AB033556.2line.fix0.1.bt2
Wrote 936 bytes to secondary EBWT file: /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/HBV_C_AB033556.2line.fix0.2.bt2
Re-opening _in1 and _in2 as input streams
Returning from Ebwt constructor
Headers:
len: 3715
bwtLen: 3716
sz: 929
bwtSz: 929
lineRate: 6
offRate: 4
offMask: 0xfffffff0
ftabChars: 10
eftabLen: 20
eftabSz: 80
ftabLen: 1048577
ftabSz: 4194308
offsLen: 233
offsSz: 932
lineSz: 64
sideSz: 64
sideBwtSz: 48
sideBwtLen: 192
numSides: 20
numLines: 20
ebwtTotLen: 1280
ebwtTotSz: 1280
color: 0
reverse: 0
Total time for call to driver() for forward index: 00:00:01
Reading reference sizes
Time reading reference sizes: 00:00:00
Calculating joined length
Writing header
Reserving space for joined string
Joining reference sequences
Time to join reference sequences: 00:00:00
Time to reverse reference sequence: 00:00:00
bmax according to bmaxDivN setting: 928
Using parameters --bmax 696 --dcv 1024
Doing ahead-of-time memory usage test
Passed! Constructing with these parameters: --bmax 696 --dcv 1024
Constructing suffix-array element generator
Building DifferenceCoverSample
Building sPrime
Building sPrimeOrder
V-Sorting samples
V-Sorting samples time: 00:00:00
Allocating rank array
Ranking v-sort output
Ranking v-sort output time: 00:00:00
Invoking Larsson-Sadakane on ranks
Invoking Larsson-Sadakane on ranks time: 00:00:00
Sanity-checking and returning
Building samples
Reserving space for 12 sample suffixes
Generating random suffixes
QSorting 12 sample offsets, eliminating duplicates
QSorting sample offsets, eliminating duplicates time: 00:00:00
Multikey QSorting 12 samples
(Using difference cover)
Multikey QSorting samples time: 00:00:00
Calculating bucket sizes
Splitting and merging
Splitting and merging time: 00:00:00
Split 1, merged 6; iterating...
Splitting and merging
Splitting and merging time: 00:00:00
Avg bucket size: 463.5 (target: 695)
Converting suffix-array elements to index image
Allocating ftab, absorbFtab
Entering Ebwt loop
Getting block 1 of 8
Reserving size (696) for bucket 1
Calculating Z arrays for bucket 1
Entering block accumulator loop for bucket 1:
bucket 1: 10%
bucket 1: 20%
bucket 1: 30%
bucket 1: 40%
bucket 1: 50%
bucket 1: 60%
bucket 1: 70%
bucket 1: 80%
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bucket 1: 100%
Sorting block of length 441 for bucket 1
(Using difference cover)
Sorting block time: 00:00:00
Returning block of 442 for bucket 1
Getting block 2 of 8
Reserving size (696) for bucket 2
Calculating Z arrays for bucket 2
Entering block accumulator loop for bucket 2:
bucket 2: 10%
bucket 2: 20%
bucket 2: 30%
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bucket 2: 100%
Sorting block of length 638 for bucket 2
(Using difference cover)
Sorting block time: 00:00:00
Returning block of 639 for bucket 2
Getting block 3 of 8
Reserving size (696) for bucket 3
Calculating Z arrays for bucket 3
Entering block accumulator loop for bucket 3:
bucket 3: 10%
bucket 3: 20%
bucket 3: 30%
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bucket 3: 50%
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bucket 3: 80%
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bucket 3: 100%
Sorting block of length 332 for bucket 3
(Using difference cover)
Sorting block time: 00:00:00
Returning block of 333 for bucket 3
Getting block 4 of 8
Reserving size (696) for bucket 4
Calculating Z arrays for bucket 4
Entering block accumulator loop for bucket 4:
bucket 4: 10%
bucket 4: 20%
bucket 4: 30%
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bucket 4: 50%
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bucket 4: 70%
bucket 4: 80%
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bucket 4: 100%
Sorting block of length 442 for bucket 4
(Using difference cover)
Sorting block time: 00:00:00
Returning block of 443 for bucket 4
Getting block 5 of 8
Reserving size (696) for bucket 5
Calculating Z arrays for bucket 5
Entering block accumulator loop for bucket 5:
bucket 5: 10%
bucket 5: 20%
bucket 5: 30%
bucket 5: 40%
bucket 5: 50%
bucket 5: 60%
bucket 5: 70%
bucket 5: 80%
bucket 5: 90%
bucket 5: 100%
Sorting block of length 425 for bucket 5
(Using difference cover)
Sorting block time: 00:00:00
Returning block of 426 for bucket 5
Getting block 6 of 8
Reserving size (696) for bucket 6
Calculating Z arrays for bucket 6
Entering block accumulator loop for bucket 6:
bucket 6: 10%
bucket 6: 20%
bucket 6: 30%
bucket 6: 40%
bucket 6: 50%
bucket 6: 60%
bucket 6: 70%
bucket 6: 80%
bucket 6: 90%
bucket 6: 100%
Sorting block of length 373 for bucket 6
(Using difference cover)
Sorting block time: 00:00:00
Returning block of 374 for bucket 6
Getting block 7 of 8
Reserving size (696) for bucket 7
Calculating Z arrays for bucket 7
Entering block accumulator loop for bucket 7:
bucket 7: 10%
bucket 7: 20%
bucket 7: 30%
bucket 7: 40%
bucket 7: 50%
bucket 7: 60%
bucket 7: 70%
bucket 7: 80%
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bucket 7: 100%
Sorting block of length 439 for bucket 7
(Using difference cover)
Sorting block time: 00:00:00
Returning block of 440 for bucket 7
Getting block 8 of 8
Reserving size (696) for bucket 8
Calculating Z arrays for bucket 8
Entering block accumulator loop for bucket 8:
bucket 8: 10%
bucket 8: 20%
bucket 8: 30%
bucket 8: 40%
bucket 8: 50%
bucket 8: 60%
bucket 8: 70%
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bucket 8: 90%
bucket 8: 100%
Sorting block of length 618 for bucket 8
(Using difference cover)
Sorting block time: 00:00:00
Returning block of 619 for bucket 8
Exited Ebwt loop
fchr[A]: 0
fchr[C]: 832
fchr[G]: 1857
fchr[T]: 2662
fchr[$]: 3715
Exiting Ebwt::buildToDisk()
Returning from initFromVector
Wrote 4195809 bytes to primary EBWT file: /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/HBV_C_AB033556.2line.fix0.rev.1.bt2
Wrote 936 bytes to secondary EBWT file: /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/HBV_C_AB033556.2line.fix0.rev.2.bt2
Re-opening _in1 and _in2 as input streams
Returning from Ebwt constructor
Headers:
len: 3715
bwtLen: 3716
sz: 929
bwtSz: 929
lineRate: 6
offRate: 4
offMask: 0xfffffff0
ftabChars: 10
eftabLen: 20
eftabSz: 80
ftabLen: 1048577
ftabSz: 4194308
offsLen: 233
offsSz: 932
lineSz: 64
sideSz: 64
sideBwtSz: 48
sideBwtLen: 192
numSides: 20
numLines: 20
ebwtTotLen: 1280
ebwtTotSz: 1280
color: 0
reverse: 1
Total time for backward call to driver() for mirror index: 00:00:00
INFO @ [2022-05-24 12:32:35 UTC]: Start to map reads to /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/HBV_C_AB033556.2line.fix0.fasta using Bowtie2.
INFO @ [2022-05-24 12:32:35 UTC]: Running Bowtie2 for paired-end read mapping
INFO @ [2022-05-24 12:32:35 UTC]: /opt/qap/bin/3rdPartyTools/bowtie2/bowtie2 --very-sensitive-local --threads 1 -x /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/HBV_C_AB033556.2line.fix0 -1 /data/data/SRR6378032_1.fastq.gz -2 /data/data/SRR6378032_2.fastq.gz -S /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/0.sam
57699 reads; of these:
57699 (100.00%) were paired; of these:
7821 (13.55%) aligned concordantly 0 times
37033 (64.18%) aligned concordantly exactly 1 time
12845 (22.26%) aligned concordantly >1 times
----
7821 pairs aligned concordantly 0 times; of these:
7622 (97.46%) aligned discordantly 1 time
----
199 pairs aligned 0 times concordantly or discordantly; of these:
398 mates make up the pairs; of these:
101 (25.38%) aligned 0 times
55 (13.82%) aligned exactly 1 time
242 (60.80%) aligned >1 times
99.91% overall alignment rate
INFO @ [2022-05-24 12:40:54 UTC]: Converting /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/0.sam to bam file
INFO @ [2022-05-24 12:40:54 UTC]: /opt/qap/bin/3rdPartyTools/samtools/samtools view -h -@ 1 -Sb /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/0.sam > /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/0.bam
INFO @ [2022-05-24 12:40:59 UTC]: Sorting bam file /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/0.bam with coordinates.
INFO @ [2022-05-24 12:40:59 UTC]: /opt/qap/bin/3rdPartyTools/samtools/samtools sort -@ 1 -O bam -T 0.PosSorted.1653396059 -o /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/0.PosSorted.bam /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/0.bam
INFO @ [2022-05-24 12:41:03 UTC]: Indexing sorted bam file.
INFO @ [2022-05-24 12:41:03 UTC]: /opt/qap/bin/3rdPartyTools/samtools/samtools index /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/0.PosSorted.bam
INFO @ [2022-05-24 12:41:03 UTC]: /opt/qap/bin/3rdPartyTools/samtools/samtools depth -aa /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/0.PosSorted.bam > /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/depth/0.depth
INFO @ [2022-05-24 12:41:05 UTC]: Rscript /opt/qap/bin/Rscripts/FindDepthInterval.R -i /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/depth/0.depth -o /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/depth/0.interval
INFO @ [2022-05-24 12:41:05 UTC]: Start to map reads to /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/HBV_C_AB033556.2line.fix0.fasta using Bowtie2.
INFO @ [2022-05-24 12:41:05 UTC]: Running Bowtie2 for paired-end read mapping
INFO @ [2022-05-24 12:41:05 UTC]: /opt/qap/bin/3rdPartyTools/bowtie2/bowtie2 --very-sensitive-local --threads 1 -x /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/HBV_C_AB033556.2line.fix0 -1 /data/data/SRR6377924_1.fastq.gz -2 /data/data/SRR6377924_2.fastq.gz -S /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/1.sam
58549 reads; of these:
58549 (100.00%) were paired; of these:
13245 (22.62%) aligned concordantly 0 times
33871 (57.85%) aligned concordantly exactly 1 time
11433 (19.53%) aligned concordantly >1 times
----
13245 pairs aligned concordantly 0 times; of these:
7533 (56.87%) aligned discordantly 1 time
----
5712 pairs aligned 0 times concordantly or discordantly; of these:
11424 mates make up the pairs; of these:
11181 (97.87%) aligned 0 times
74 (0.65%) aligned exactly 1 time
169 (1.48%) aligned >1 times
90.45% overall alignment rate
INFO @ [2022-05-24 12:48:48 UTC]: Converting /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/1.sam to bam file
INFO @ [2022-05-24 12:48:48 UTC]: /opt/qap/bin/3rdPartyTools/samtools/samtools view -h -@ 1 -Sb /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/1.sam > /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/1.bam
INFO @ [2022-05-24 12:48:53 UTC]: Sorting bam file /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/1.bam with coordinates.
INFO @ [2022-05-24 12:48:53 UTC]: /opt/qap/bin/3rdPartyTools/samtools/samtools sort -@ 1 -O bam -T 1.PosSorted.1653396533 -o /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/1.PosSorted.bam /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/1.bam
INFO @ [2022-05-24 12:48:57 UTC]: Indexing sorted bam file.
INFO @ [2022-05-24 12:48:57 UTC]: /opt/qap/bin/3rdPartyTools/samtools/samtools index /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/1.PosSorted.bam
INFO @ [2022-05-24 12:48:57 UTC]: /opt/qap/bin/3rdPartyTools/samtools/samtools depth -aa /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/1.PosSorted.bam > /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/depth/1.depth
INFO @ [2022-05-24 12:48:58 UTC]: Rscript /opt/qap/bin/Rscripts/FindDepthInterval.R -i /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/depth/1.depth -o /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/depth/1.interval
INFO @ [2022-05-24 12:48:59 UTC]: Start to map reads to /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/HBV_C_AB033556.2line.fix0.fasta using Bowtie2.
INFO @ [2022-05-24 12:48:59 UTC]: Running Bowtie2 for paired-end read mapping
INFO @ [2022-05-24 12:48:59 UTC]: /opt/qap/bin/3rdPartyTools/bowtie2/bowtie2 --very-sensitive-local --threads 1 -x /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/HBV_C_AB033556.2line.fix0 -1 /data/data/SRR6377925_1.fastq.gz -2 /data/data/SRR6377925_2.fastq.gz -S /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/2.sam
55473 reads; of these:
55473 (100.00%) were paired; of these:
8817 (15.89%) aligned concordantly 0 times
32750 (59.04%) aligned concordantly exactly 1 time
13906 (25.07%) aligned concordantly >1 times
----
8817 pairs aligned concordantly 0 times; of these:
6382 (72.38%) aligned discordantly 1 time
----
2435 pairs aligned 0 times concordantly or discordantly; of these:
4870 mates make up the pairs; of these:
3810 (78.23%) aligned 0 times
935 (19.20%) aligned exactly 1 time
125 (2.57%) aligned >1 times
96.57% overall alignment rate
INFO @ [2022-05-24 12:55:21 UTC]: Converting /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/2.sam to bam file
INFO @ [2022-05-24 12:55:21 UTC]: /opt/qap/bin/3rdPartyTools/samtools/samtools view -h -@ 1 -Sb /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/2.sam > /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/2.bam
INFO @ [2022-05-24 12:55:26 UTC]: Sorting bam file /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/2.bam with coordinates.
INFO @ [2022-05-24 12:55:26 UTC]: /opt/qap/bin/3rdPartyTools/samtools/samtools sort -@ 1 -O bam -T 2.PosSorted.1653396926 -o /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/2.PosSorted.bam /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/2.bam
INFO @ [2022-05-24 12:55:30 UTC]: Indexing sorted bam file.
INFO @ [2022-05-24 12:55:30 UTC]: /opt/qap/bin/3rdPartyTools/samtools/samtools index /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/2.PosSorted.bam
INFO @ [2022-05-24 12:55:30 UTC]: /opt/qap/bin/3rdPartyTools/samtools/samtools depth -aa /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/bwa/2.PosSorted.bam > /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/depth/2.depth
INFO @ [2022-05-24 12:55:31 UTC]: Rscript /opt/qap/bin/Rscripts/FindDepthInterval.R -i /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/depth/2.depth -o /qap_Results_for_FixCircRef_20220524_12h32m34s/tmp/depth/2.interval
INFO @ [2022-05-24 12:55:32 UTC]: Program completed!

we limit our scope to applications of NGS where aligned reads are considered to be a population sample. In this definition of deep sequencing, reads aligning to a given genomic position are each assumed to originate from an individual replicon, revealing a snapshot of the population’s genetic diversity.

Sequencing of populations, on the other hand, has the drawback that unless variants co-occur within a read length or read pair, it is difficult to reconstruct individual haplotypes and perform phylogenetic analysis.

This ability to detect low frequency variants is an important feature of deep sequencing, for example in the context of drug resistance.
深度测序对用于发现低频突变从而来分析耐药性。

Sequencing of populations, on the other hand, has the drawback that unless variants co-occur within a read length or read pair, it is difficult to reconstruct individual haplotypes and perform phylogenetic analysis.
深度测序对于构建单倍型和进化分析来说 就很困难。

either by simply considering physical linkage of SNVs within individual reads, or by using overlapping reads to reconstruct longer genome fragments, termed ‘haplotypes’.