This package was created in order to increase the sensitivity of EML4-ALK detection from commercially available NGS products such as the AVENIO (Roche) pipeline.
Paired-end sequencing of cfDNA generated BAM files can be used as input to discover EML4-ALK variants. This package was developed using position deduplicated BAM files generated with the AVENIO Oncology Analysis Software. These files are made using the AVENIO ctDNA surveillance kit and Illumina Nextseq 500 sequencing. This is a targeted hybridization NGS approach and includes ALK-specific but not EML4-specific probes.
The package includes six functions.
The output of the first function, EML4_ALK_detection()
, is used to determine
whether EML4-ALK is detected and serves as input for the next four exploratory
functions characterizing the EML4-ALK variant. The last function
EML4_ALK_analysis()
combines the output of the exploratory functions.
To serve as examples, this package includes BAM files representing the EML4-ALK positive cell line H3122 and the EML4-ALK negative cell line, HCC827.
Use Bioconductor to install the most recent version of DNAfusion
BAM files from the cell lines, H3122 and HCC827, are included in the package and can be used as examples to explore the functions.
EML4_ALK_detection()
This function looks for EML4-ALK mate pair reads in the BAM file.
Input: \[\\[0.1in]\]
file
The name of the file which the data are to be read from.
\[\\[0.1in]\]
genome
character representing the reference genome.
Can either be "hg38" or "hg19".
Default = "hg38".
\[\\[0.1in]\]
mates
integer, the minimum number EML4-ALK mate pairs needed to be
detected in order to call a variant. Default = 2.
\[\\[0.1in]\]
Output:
A GAlignments
object with soft-clipped reads representing
EML4-ALK is returned. If no EML4-ALK is detected the the GAlignments
is empty.
Examples:
H3122_result <- EML4_ALK_detection(file = H3122_bam,
genome = "hg38",
mates = 2)
head(H3122_result)
#> GAlignments object with 6 alignments and 2 metadata columns:
#> seqnames strand cigar qwidth start end width
#> <Rle> <Rle> <character> <integer> <integer> <integer> <integer>
#> [1] chr2 + 94M2S 96 42299657 42299750 94
#> [2] chr2 + 94M2S 96 42299657 42299750 94
#> [3] chr2 + 94M2S 96 42299657 42299750 94
#> [4] chr2 + 94M2S 96 42299657 42299750 94
#> [5] chr2 + 94M2S 96 42299657 42299750 94
#> [6] chr2 + 94M2S 96 42299657 42299750 94
#> njunc | mpos seq
#> <integer> | <integer> <DNAStringSet>
#> [1] 0 | 29223691 TTGCTTCTTT...GCAGTGGTCT
#> [2] 0 | 29223375 TTGCTTCTTT...GCAGTGGTCT
#> [3] 0 | 29223479 TTGCTTCTTT...GCAGTGGTCT
#> [4] 0 | 29223686 TTGCTTCTTT...GCAGTGGTCT
#> [5] 0 | 29223636 TTGCTTCTTT...GCAGTGGTCT
#> [6] 0 | 29223687 TTGCTTCTTT...GCAGTGGTCT
#> -------
#> seqinfo: 455 sequences from an unspecified genome
HCC827_result <- EML4_ALK_detection(file = HCC827_bam,
genome = "hg38",
mates = 2)
HCC827_result
#> GAlignments object with 0 alignments and 0 metadata columns:
#> seqnames strand cigar qwidth start end width
#> <Rle> <Rle> <character> <integer> <integer> <integer> <integer>
#> njunc
#> <integer>
#> -------
#> seqinfo: no sequences
EML4_sequence()
This function identifies the basepairs leading up to the EML4 breakpoint.
Input: \[\\[0.1in]\]
reads
GAlignments object returned by EML4_ALK_detection().
\[\\[0.1in]\]
basepairs
integer, number of basepairs identified from the EML4-ALK fusion.
Default = 20.
\[\\[0.1in]\]
Output:
If EML4-ALK is detected, returns a table
of identified
EML4 basepairs with the number of corresponding reads for each sequence.
If no EML4-ALK is detected “No EML4-ALK was detected” is returned.
Examples:
ALK_sequence()
This function identifies the basepairs following the ALK breakpoint.
Input: \[\\[0.1in]\]
reads
GAlignments object returned by EML4_ALK_detection().
\[\\[0.1in]\]
basepairs
integer, number of basepairs identified from the EML4-ALK fusion.
Default = 20.
\[\\[0.1in]\]
Output:
If EML4-ALK is detected, returns a table
of identified
ALK basepairs with the number of corresponding reads for each sequence.
If no EML4-ALK is detected “No EML4-ALK was detected” is returned.
Examples:
ALK_sequence(H3122_result, basepairs = 20)
#> ALK_seq
#> CAGAATTTTAGCTTTGCAAT CGGAATTTTAGCTTTGCATT CGGATTTTTAGCTTT
#> 1 1 1
#> CGGATTTTTAGCTTTTCATT CT CTG
#> 2 8 3
#> CTGA CTGAA CTGAT
#> 11 1 16
#> CTGATTTT CTGATTTTT CTGATTTTTA
#> 5 6 3
#> CTGATTTTTAG CTGATTTTTAGATTTGCATT CTGATTTTTAGC
#> 3 1 14
#> CTGATTTTTAGCT CTGATTTTTAGCTT CTGATTTTTAGCTTT
#> 10 10 3
#> CTGATTTTTAGCTTTG CTGATTTTTAGCTTTGC CTGATTTTTAGCTTTGCA
#> 4 7 8
#> CTGATTTTTAGCTTTGCAAT CTGATTTTTAGCTTTGCAT CTGATTTTTAGCTTTGCATT
#> 1 1 71
#> CTGATTTTTAGCTTTT CTGATTTTTAGCTTTTCATA CTGATTTTTAT
#> 1 1 1
#> CTGATTTTTATCTTTG CTGATTTTTATCTTTGCATT CTGATTTTTATCTTTTGATT
#> 2 2 1
#> CTGTGTTTTAGATTTGCATT CTGTTTTTTATCTTTGCAAT CTTATTTTTATCTTTGCATT
#> 1 1 1
#> TTAGCTTTG
#> 1
ALK_sequence(HCC827_result, basepairs = 20)
#> [1] "No EML4-ALK was detected"
break_position()
This function identifies the genomic position in EML4 where the breakpoint has happened.
Input: \[\\[0.1in]\]
reads
GAlignments object returned by EML4_ALK_detection().
\[\\[0.1in]\]
Output:
If EML4-ALK is detected, returns a table
of genomic positions
with the number of corresponding reads for each sequence.
If no EML4-ALK is detected “No EML4-ALK was detected” is returned.
Examples:
break_position_depth()
This function identifies the read depth at the basepair before the breakpoint in EML4.
Input: \[\\[0.1in]\]
file
The name of the file which the data are to be read from.
\[\\[0.1in]\]
reads
GAlignments returned by EML4_ALK_detection().
\[\\[0.1in]\]
Output:
If EML4-ALK is detected a single integer
corresponding
to the read depth at the breakpoint is returned.
If no EML4-ALK is detected “No EML4-ALK was detected” is returned.
Examples:
EML4_ALK_analysis()
This functions collects the results from the other functions of the package.
Input: \[\\[0.1in]\]
file
The name of the file which the data are to be read from.
\[\\[0.1in]\]
genome
character representing the reference genome.
Can be either "hg38" or "hg19".
Default = "hg38".
\[\\[0.1in]\]
mates
integer, the minimum number EML4-ALK mate pairs needed to be detected in
order to call a variant. Default = 2.
\[\\[0.1in]\]
basepairs
integer, number of basepairs identified from the EML4-ALK fusion.
Default = 20.
\[\\[0.1in]\]
Output:
A list
object with
clipped_reads corresponding to EML4_ALK_detection()
,
last_EML4 corresponding to EML4_sequence()
,
first_ALK corresponding to ALK_sequence()
,
breakpoint corresponding to break_position()
,
and read_depth corresponding to break_position_depth()
.
If no EML4-ALK is detected an empty GAlignments
is returned.
Examples:
H3122_results <- EML4_ALK_analysis(file = H3122_bam,
genome = "hg38",
mates = 2,
basepairs = 20)
HCC827_results <- EML4_ALK_analysis(file = HCC827_bam,
genome = "hg38",
mates = 2,
basepairs = 20)
head(H3122_results$clipped_reads)
#> GAlignments object with 6 alignments and 2 metadata columns:
#> seqnames strand cigar qwidth start end width
#> <Rle> <Rle> <character> <integer> <integer> <integer> <integer>
#> [1] chr2 + 94M2S 96 42299657 42299750 94
#> [2] chr2 + 94M2S 96 42299657 42299750 94
#> [3] chr2 + 94M2S 96 42299657 42299750 94
#> [4] chr2 + 94M2S 96 42299657 42299750 94
#> [5] chr2 + 94M2S 96 42299657 42299750 94
#> [6] chr2 + 94M2S 96 42299657 42299750 94
#> njunc | mpos seq
#> <integer> | <integer> <DNAStringSet>
#> [1] 0 | 29223691 TTGCTTCTTT...GCAGTGGTCT
#> [2] 0 | 29223375 TTGCTTCTTT...GCAGTGGTCT
#> [3] 0 | 29223479 TTGCTTCTTT...GCAGTGGTCT
#> [4] 0 | 29223686 TTGCTTCTTT...GCAGTGGTCT
#> [5] 0 | 29223636 TTGCTTCTTT...GCAGTGGTCT
#> [6] 0 | 29223687 TTGCTTCTTT...GCAGTGGTCT
#> -------
#> seqinfo: 455 sequences from an unspecified genome
H3122_results$last_EML4
#> EML4_seq
#> CCAGGCTGGAGTGCAGTGGT GGAGTGCAGTGGTGTGATTT TCAGGCTGGAGTGCAGTGGT
#> 201 1 1
H3122_results$first_ALK
#> ALK_seq
#> CAGAATTTTAGCTTTGCAAT CGGAATTTTAGCTTTGCATT CGGATTTTTAGCTTT
#> 1 1 1
#> CGGATTTTTAGCTTTTCATT CT CTG
#> 2 8 3
#> CTGA CTGAA CTGAT
#> 11 1 16
#> CTGATTTT CTGATTTTT CTGATTTTTA
#> 5 6 3
#> CTGATTTTTAG CTGATTTTTAGATTTGCATT CTGATTTTTAGC
#> 3 1 14
#> CTGATTTTTAGCT CTGATTTTTAGCTT CTGATTTTTAGCTTT
#> 10 10 3
#> CTGATTTTTAGCTTTG CTGATTTTTAGCTTTGC CTGATTTTTAGCTTTGCA
#> 4 7 8
#> CTGATTTTTAGCTTTGCAAT CTGATTTTTAGCTTTGCAT CTGATTTTTAGCTTTGCATT
#> 1 1 71
#> CTGATTTTTAGCTTTT CTGATTTTTAGCTTTTCATA CTGATTTTTAT
#> 1 1 1
#> CTGATTTTTATCTTTG CTGATTTTTATCTTTGCATT CTGATTTTTATCTTTTGATT
#> 2 2 1
#> CTGTGTTTTAGATTTGCATT CTGTTTTTTATCTTTGCAAT CTTATTTTTATCTTTGCATT
#> 1 1 1
#> TTAGCTTTG
#> 1
H3122_results$breakpoint
#> break_pos
#> 42299750 42299757
#> 202 1
H3122_results$read_depth
#> [1] 251
HCC827_results
#> GAlignments object with 0 alignments and 0 metadata columns:
#> seqnames strand cigar qwidth start end width
#> <Rle> <Rle> <character> <integer> <integer> <integer> <integer>
#> njunc
#> <integer>
#> -------
#> seqinfo: no sequences
#> ─ Session info ───────────────────────────────────────────────────────────────
#> setting value
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#> ui X11
#> language (EN)
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#> tz America/New_York
#> date 2022-11-01
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#> devtools * 2.4.5 2022-10-11 [2] CRAN (R 4.2.1)
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